J.1747 0285.2009.00940.x

ª 2010 John Wiley & Sons A/S
Chem Biol Drug Des 2010: 75: 237–256 doi: 10.1111/j.1747-0285.2009.00940.x

Review Article

NMR-Based Screening of Membrane Protein
Ligands

Naveena Yanamala1,†, Arpana Dutta1,†, channels or transporters. Among these, the G-protein-coupled
Barbara Beck2, Bart van Fleet2, Kelly Hay2, receptors (GPCRs) are the largest group of drug targets because
Ahmad Yazbak3, Rieko Ishima1, Alexander of their important role in mediating communication between the
Doemling2,* and Judith Klein-Seetharaman1,* inside and outside of the cell in response to an enormous variety
of different ligands, ranging from small proteins and peptides to
1
Departments of Structural Biology, 2Pharmaceutical Sciences and small organic molecules, ions and even light. These ligands can
Chemistry, University of Pittsburgh, Pittsburgh, PA15260, USA be hormones, odorants, neurotransmitters or other functional clas-
3 ses of biologically active compounds. Despite the importance of
Synthatex, Shefa-Amr Industrial Park, PO Box 437 Shefa Amr
20200, Israel membrane proteins as drug targets, they have not been very ame-
*Corresponding author: Judith Klein-Seetharaman, jks33@pitt.edu; nable to structure-based drug design. This is because the hydro-
Alexander Doemling, asd30@pitt.edu phobic nature of their transmembrane regions hampers
† crystallization as well as NMR-spectroscopic analysis. Progress in
These authors contributed equally to this manuscript.
membrane protein structure determination by NMR is steadily
Membrane proteins pose problems for the appli- being made, with some recent spectacular breakthrough achieve-
cation of NMR-based ligand-screening methods ments in the sizes of protein structures obtained for both b-barrel
because of the need to maintain the proteins in a membrane proteins (1,2) and a-helical proteins (3). Because the
membrane mimetic environment such as detergent structure determination of membrane proteins involves extensive
micelles: they add to the molecular weight of the detergent screening and the selection of suitable buffer condi-
protein, increase the viscosity of the solution, inter- tions, it is not a routine application. Thus, NMR structure-based
act with ligands non-speciﬁcally, overlap with pro- drug design involving membrane protein targets still remains a
tein signals, modulate protein dynamics and
future goal. However, this does not preclude the application of
conformational exchange and compromise sensitiv-
ity by adding highly intense background signals. In
NMR techniques to membrane protein drug discovery. In particular,
this article, we discuss the special considerations NMR spectroscopy can yield high-quality ligand-binding information
arising from these problems when conducting NMR- even in the absence of the structures of the targets. This article
based ligand-binding studies with membrane pro- will explore the applicability of different NMR-spectroscopic
teins. While the use of 13C and 15N isotopes is approaches to the study of ligand–membrane protein interactions
becoming increasingly feasible, 19F and 1H NMR- from a fundamental perspective keeping in mind their potential
based approaches are currently the most widely use in drug discovery.
explored. By using suitable NMR parameter selec-
tion schemes independent of or exploiting the pres-
This review is organized as follows. First, in 'NMR-based
ence of detergent, 1H-based approaches require
approaches to drug screening', we will briefly review different
least effort in sample preparation because of the
high sensitivity and natural abundance of 1H in NMR-based approaches to the study of ligand binding to soluble
both, ligand and protein. On the other hand, the 19F proteins. In 'Challenges in membrane protein NMR spectroscopy',
nucleus provides an ideal NMR probe because of its we will highlight the applicability and special considerations for
similarly high sensitivity to that of 1H and the lack NMR-based approaches in the context of membrane protein stud-
of natural 19F background in biologic systems. ies. '1H NMR-based approaches for membrane proteins' describes
1
Despite its potential, the use of NMR spectroscopy H NMR-spectroscopic approaches, and '19F NMR-based
is highly underdeveloped in the area of drug discov- approaches' provides an overview of 19F NMR-spectroscopic
ery for membrane proteins.
approaches. 'Comparison of 1H and 19F-NMR-based versus conven-
tional screening of membrane proteins' will discuss the advanta-
Received 29 July 2009, revised 30 November 2009 and accepted for
publication 30 November 2009 ges and disadvantages of 1H and 19F NMR-based screening
methods when compared to other high-throughput screening (HTS)
approaches. 'Synthesis of 19F containing small molecule com-
Membrane proteins are encoded by up to 30% of typical genomes pounds' will describe the practical aspects of obtaining 19F con-
and constitute the most important class of drug targets: more taining small molecule compound libraries. Finally, we will
than 60% of current drugs are targeting membrane receptors, conclude with 'Summary and outlook'.

237

Yanamala et al.

NMR-based approaches to drug Chemical shift changes and line broadening are parameters that
screening can be used for screening even if resonance assignment is not fea-
sible. More information, however, can be obtained when signals
Although NMR-based screening is only one of many screening are assigned. In that case, the changes in chemical shift or broad-
tools in drug discovery, its simplicity, wide range of application ness of lines can be used to generate testable hypotheses on what
(including protein–protein and protein–nucleic acid interactions) are the residues in contact with the ligand, or which are allosteri-
and superior ability to detect weakly bound molecules have cally modulated by ligand binding.
attracted much attention. Nuclear magnetic resonance allows the
measurement of multiple parameters at different levels of com- Even more information can be extracted, if the protein structure is
plexity and information content. Thus, NMR-based methods differ known. In particular, the pioneering work of Fesik at Abbott Laborato-
significantly from one another as a result of the particular ries (Illinois, USA) opened a new field in the area of fast and
approach used. Excellent reviews of different NMR-screening efficient drug discovery, a technique coined structure–activity rela-
methods are provided for example in (4–7). Here, we briefly tionships by nuclear magnetic resonance ('SAR by NMR') (12). The
review the different methods that have been mainly employed Abbott group uses 2-dimensional 1H,15N-HSQC spectra to screen
with soluble proteins to provide an idea of the scope of small molecular weight compounds for binding to 15N-labeled pro-
approaches with potential or realized applicability to membrane teins of determined structures. Structure–activity relationships by
protein ligand screening. Fundamentally, two types of experiments NMR locates the binding site for the ligand on a protein's surface
can be distinguished in NMR-based screening approaches: one to because the resonances have been assigned prior to ligand screen-
detect protein signals (Screening of ligands by detecting targeting, and the structure of the protein is known. Comparing the struc-
protein signals) and the other to detect ligand signals (Screening tures of compounds that bind to the same site on a protein provides
of ligands by detecting ligand signals). There are also specialized information about the functional groups involved in ligand binding
improvements in technology to increase throughput or to study and can guide the synthesis of lead compounds by medicinal chemis-
particular types of ligands such as those that disrupt protein–pro- try. This technique is restricted, however, to protein sizes of less than
tein interactions (Other NMR-based screenings). Because sensitivity 30 000 D because of limitation by the molecular rotational
of the observed NMR signals in the ligand–protein interacting sys- correlation times leading to broad NMR lines for larger proteins.
tems depends on binding affinity, the estimation of the ligand dis- Many compounds have been discovered by this technique (13), and
sociation constant (or binding constant) is also described several compounds emerged in human clinical trials (14).
(Determination of ligand-binding constants by NMR), before we
end with a Summary. In cases where protein signals have not been or cannot be identi-
fied, other lead optimization methods such as Inter-Ligand NOE
Screening of ligands by detecting target- (ILOE) and ILOE for Pharmacophore Mapping (INPHARMA) can be
protein signals used to detect protein-mediated ligand–ligand interactions by
In protein-detection based screening, the identification of ligand detecting ligand signals (15,16). The principle of these methods is
binding is based on changes in NMR signals arising from proteins, based on two ligands binding to the same protein. ILOE is used to
typically in one-dimensional 1H spectra or two-dimensional 1H,15N- identify pairs of small molecules that bind to adjacent sites on the
heteronuclear single quantum correlation (HSQC) spectra. Because surface of the target protein (15). In contrast to the ILOE's detection
of the large number of peaks in proteins, two-dimensional experi- of simultaneous ligand binding at two different but proximal sites
ments will afford better resolution of signals but require that the in the protein (15), the INPHARMA technique is specialized to iden-
protein is labeled. The longer data acquisition times for higher tify ligands that compete for the same ligand-binding site (16). The
dimensional spectra are also a drawback, especially when screening idea in the ILOE approach is similar to the SAR by NMR approach
larger numbers of ligands. Recent efforts are therefore aimed at in that the occupation of proximal but initially independent ligand-
decreasing the acquisition time, including 'SOFAST- HMQC' or binding pockets can be combined with a single ligand targeting
'Ultra-fast experiments' (8,9). both pockets to obtain higher affinity ligands. In the INPHARMA
approach, the two ligands are never close in space or bound to the
Binding information can be obtained for one- or two-dimensional protein simultaneously, but rather the observed NOEs are mediated
spectra regardless of whether the signals are assigned or not by sim- by spin diffusion via the protons on the protein. The advantage of
ply recording if signals show altered chemical shifts or line broad- the ILOE and INPHARMA methods is that assignment and structure
ness, and many screening programs are based on this approach (6). of the protein do not need to be known for lead optimization.
Broadening of the NMR signals is observed when the exchange rate
(defined by the population weighed on ⁄ off-rate of the ligand) is simi-
lar to the difference in chemical shifts between the free and bound Screening of ligands by detecting ligand signals
forms (10). Changes in signal positions are only observed when the Protein-detection-based methods suffer from the general drawback
exchange rate is slow, i.e., the ligand binds tightly. Broadening and that NMR lines become broader with the increased size of the mol-
changes in chemical shifts of signals upon ligand binding are ecule under study. This makes it desirable to measure the ligand
determined by the differences in chemical shifts, the relative instead of the protein, because ligands are typically small mole-
protein ⁄ ligand molar ratio and the on ⁄ off-rate of the ligand. For an cules and will give rise to much sharper and more intense signals.
in-depth discussion of the different regimes, see (7,11). Thus, NMR-based screening has often made use of detecting

238 Chem Biol Drug Des 2010; 75: 237–256

NMR-Based Screening

signals of the ligands that interact with target proteins. There are water-ligand molecules that are located on the target-protein sur-
multiple ways by which ligand signals can carry information on pro- face, the NOE is negative (19,20).
tein binding, and these can be detected by classical NMR parame-
ters such as chemical shift and relaxation. Excellent overviews are
provided for example in (6,7). Other NMR-based screenings
To improve the HTS capabilities of NMR-based approaches, target-
A popular approach for ligand screening is based on the transferred immobilized NMR screening (TINS) has been proposed (21). Here,
NOE (trNOE) mechanism. Proton-proton cross-relaxation exhibits the protein target is immobilized on a gel-based solid support. This
positive NOE peaks for small molecules alone (MW < 2000 D) that is associated with several potential advantages: the target does not
undergo fast molecular tumbling, whereas negative NOE peaks are need to be soluble or even be a protein; the quantity of required
observed when the molecular tumbling becomes slow by forming a target is reduced, as a single sample of the target is sufficient for
complex with the target protein. Because ligands are at equilibrium a flow-through screen. With TINS, compound libraries can be
between the free form and bound to the target protein, the NOE screened much faster than using a traditional NMR sample in solu-
intensity that is encoded during the bound state is transferred by tion.
the exchange and observed at the free ligand signal position. Other
methods that are based on the cross-relaxation mechanism include In addition to screening, the binding of ligands to single proteins
saturation transfer-difference (STD) experiments, Water-LOGSY, such as enzymes or receptors, it is becoming increasingly important
cross-saturation, transient trNOE and NOE pumping (7). to investigate ligands interfering with protein–protein interactions,
as the importance of protein–protein interactions as targets
Saturation transfer-difference (STD) experiments detect inter-molec- increases. A fast and information-rich NMR-based technique to
ular magnetization transfer by taking the difference of two NMR screen antagonists of protein–protein interactions has recently been
spectra recorded with and without saturation of protein signals described by Holak et al. (22). This experiment has been coined
(17). The mechanism of this approach is based on rapid proton spin NMR-based Antagonist Induced Dissociation Assay (AIDA) for the
diffusion in proteins: in large proteins, once a part of the protein validation of inhibitors acting on protein–protein interactions
signal is irradiated, the saturation is transferred to the entire pro- (Figure 1). Antagonist Induced Dissociation Assay detects signals
tein within 0.1 seconds (18). The application of STD to membrane appearing upon the dissociation of the target-protein complexes.
proteins is discussed in 1H NMR-based approaches for membrane The approach requires a large protein fragment (larger than 30 kDa)
proteins. to bind to a small reporter protein (less than 20 kDa). This method-
ology has been successfully used to discover novel p53 ⁄ mdm2
Another mechanism for communication between ligand and protein antagonists (23). A cost of goods saving 1D AIDA technique has
is via water molecules (19,20). This approach is based on the obser- been described recently as well, in which tryptophan resonances
vation that ligands are often hydrated when bound to protein, or are used as reporters for ligand-binding events because of their
specifically mediate the interactions between ligand and protein via separation from most other signals in proton NMR spectra of
hydrogen bonds. Thus, by excitation of water, ligand and protein proteins (24).
sense their proximity. This mechanism is the basis for the
Water-Ligand Observation with Gradient Spectroscopy (Water- In contrast to the earlier mentioned in vitro assays, there are also
LOGSY) technique that detects water-ligand NOE transfer. For efforts to conduct screening in vivo. The approach is called small

A

B

C

D

Figure 1: Schematic outlining the principle of the AIDA technique to screen for ligands. Here, AIDA was used to discover antagonists of
the protein–protein interaction between p53 and mdm2. Left: structure (pdb identifier 1YCR) of the complex between p53 (blue helix) and
mdm2 (yellow surface). Nuclear magnetic resonance screening of chemical compounds schematically drawn in the middle yields the 1D AIDA
proton NMR spectra of the p53 ⁄ mdm2 complex on the right. Spectra labeled A–D exhibit signals from p53 in the presence of augmenting
concentrations of an antagonist. (A) no antagonist added (W23 is buried and does not give a signal). (B–D) increasing concentrations of an
antagonist are added and more and more complex dissociates. This can be seen by the increase in the intensity of the W23 peak.

Chem Biol Drug Des 2010; 75: 237–256 239

Yanamala et al.

molecule interactor library (SMILI)-NMR (25). This method records the experimental salicylate binding data, while in-depth studies
NMR signals of a protein that is over-expressed in Escherichia coli reveal the multiple site binding modes of this ligand (30).
and elucidates changes in signal positions and broadening upon
ligand interactions (26). The in-cell NMR approach has also been For sub-micromolar affinity ligands where the free ligand peak is
applied to observe and disrupt protein–protein interactions, coined unaffected by the bound state, reporter ligands can be used for
Structural Interactions (STINT) NMR (27). The advantages of the in screening (31). In this approach, the known ligand is prebound and
vivo studies are the detection of signals of unpurified proteins and the new ligands in the screen are tested for their ability to displace
information for more biologically relevant in vivo protein structures the bound ligand. For example, in the case of BSA, this approach
and interactions. Expansion of in vivo ligand-binding studies to has been taken to study tryptophan binding: complementary to the
1
mammalian cells has recently enhanced the relevance and informa- H NMR studies of BSA described earlier, 19F NMR-based studies
tion content of the technique (28). of L-5-tryptophan (32) and L-6-tryptophan (33) binding to BSA have
been carried out. The extreme sensitivity of the 19F chemical shift
resulted in the observation of two distinct peaks, indicating the
Determination of ligand-binding constants by presence of multiple tryptophan binding sites, a low-affinity and a
NMR high-affinity binding site. Competition with non-fluorinated trypto-
Typically, ligand-protein titration is conducted by observing protein phan can be used to establish relative affinities of these ligands
signals to determine ligand association ⁄ dissociation constants. First, with respect to tryptophan at both sites. Thus, while the 19F
based on the equation of the dissociation constant, when the disso- approach – unlike the 1H approach – is restricted to ligands that
ciation constant of the ligand is around 1 lM (tentatively defining bind at the same sites as 19F-containing ligands do, the 19F NMR
moderate binding), approximately 99% of the protein binds the studies proved useful in revealing an additional tryptophan-binding
ligand at 0.1 mM protein concentration with almost equal amount site that went undetected with 1H NMR, showing the complemen-
of the ligand. Upon varying the ligand concentration, the population tary nature of the approaches.
of the bound form is consequently changed. Therefore, the titration
curve is generated by plotting changes in the peak positions or sig-
nal intensities to determine the dissociation constant. Next, when Summary
the dissociation is above 1 lM (tentatively defining as weak bind- In summary, NMR techniques for drug discovery are high-content
ing), larger amounts of the ligand is required to saturate the protein methods: they potentially provide binding information, the location
signals to the bound form. Because of limitations in ligand solubility of the binding site and the conformation of the bound ligand.
or appearing of non-specific interactions at high ligand concentra- Nuclear magnetic resonance can also supply structural information
tions, it is possible that the dissociation constant is not well deter- that enables the docking of the ligand to the protein's binding
mined by NMR for very weakly interacting systems. Finally, when pocket. In addition, NMR provides very valuable information about
the dissociation constant is significantly lower than lM, such as nM the general behavior of the ligands that other HTS methods do not
(strong binding), the titration curve becomes so sharp that an reveal, including solubility, binding behavior (promiscuous ligands),
accurate dissociation constant is not obtained. precipitation potential and aggregation. Because NMR-based
screening is sensitive toward finding medium-affinity to low-affinity
Determining ligand affinity using ligand signals is not straightfor- ligands, the approach can also serve as an effective prescreening
ward. When the binding is strong, the ligand-saturated point is dif- tool for subsequent assay-based HTS. Thus, NMR-based screening
ficult to detect because ligand signals become broadened upon for small molecular weight drugs is now well established in indus-
binding to the protein. When the binding is weak, interaction is try and can be used complementary to HTS methods and computa-
better detected using the STD technique and other experiments tional screening methods.
described earlier. However, it is difficult to determine the dissocia-
tion constant accurately because other rate constants, such as
cross-relaxation rates, are involved in such experiments. Challenges in membrane protein NMR
spectroscopy
These issues are illustrated by the case studies of different ligands
binding to the model protein bovine serum albumin (BSA). Bovine While 1H NMR-based methods to study ligand binding can be car-
serum albumin binds a variety of different ligands including moder- ried out with unlabeled protein, more sophisticated applications of
ate-affinity (lM), high-affinity (nM) and low and ⁄ or varying affinity NMR-spectroscopic techniques such as SAR by NMR require label-
multisite binding ligands. For example, L-tryptophan is a moderate- ing, typically the biosynthetic introduction of 13C and 15N nuclei.
affinity ligand, while naproxen is a high-affinity ligand, and salicy- However, many proteins cannot be successfully expressed in E. coli
late has been proposed to bind to 76 binding sites in total (29). A or Pichia pastoris that make uniform 13C, 15N labeling affordable.
systematic review of 1H NMR spectroscopy of these different types When proteins need to be expressed in mammalian or insect cell
of ligands and combinations thereof (30) has yielded the following lines to obtain them in functional form, uniform labeling becomes
conclusions: when measuring 1H NMR chemical shifts and line prohibitively expensive when the protein expression levels are not
widths, titrations of different ligand ⁄ protein ratios are needed to unusually high. In such cases, specific 15N-labeled and ⁄ or
13
obtain an accurate binding constant. Particularly, careful measure- C-labeled amino acids are introduced (34–36). Such proteins are
ments and analyses have to be carried out for multisite ligands: a not amenable to structure determination by NMR spectroscopy.
wrong 1:1 binding model can provide a visually acceptable fit to Mammalian membrane proteins often belong to this group, e.g.,


NMR-Based Screening

when they are glycosylated or otherwise post-translationally modi- proteins, they typically do not bind ligands in functional form
fied in their native form and require the mammalian or insect cell excluding such systems from NMR-based ligand screening
machinery for proper folding. approaches.

NMR signal assignment requires well-resolved mono-disperse spec- The reason for the difficulties in obtaining membrane protein struc-
tra as a prerequisite, in which a large number of the NMR-active tures by NMR is largely based on the fact that NMR signals
nuclei in the sample are visible and resolved from each other, and become broader as the molecular mass increases, leading to the
the signal intensity for different peaks is as uniform as possible. reduction in sensitivity of NMR experiments. Because membrane
This in part is the reason for the limit in size of biomolecules that proteins are studied under conditions surrounded by micelles
can be studied, but poor quality spectra can also arise from sys- formed by the detergents, the apparent molecular mass becomes
tems that are dynamic and ⁄ or prone to aggregation even when the larger than the protein molecular weight. Also, when membrane
size of the monomeric unit is relatively small, depending on the proteins form biologically functional or non-functional oligomers, the
propensity of the proteins and choice of detergents. Thus, it is criti- apparent molecular mass, including the surrounding deter-
cal to choose suitable detergents for each membrane protein. After gent ⁄ micelles, results in further broadening of NMR signals. Thus,
or complementary to light-scattering experiments, 1H,15N-HSQC several efforts are underway to detect protein NMR signals of large
spectra are typically recorded to screen for detergents and other proteins, which are useful for drug screening and ⁄ or signal assign-
conditions, such as salt concentration and pH, under which reason- ment purposes: fast experiments, TROSY methods and various iso-
able NMR spectra can be obtained. Recent developments in micro- tope labeling techniques. TROSY in particular has been crucial in all
coil NMR technology have the potential to make the screening of a of the recent determinations of membrane protein structures but
large number of different detergents for their suitability to support requires deuteration. Efforts to detect NMR signals in shorter time,
NMR studies more feasible (37). such as 'SOFAST- HMQC' or 'Ultra-fast experiments' may prove use-
ful for drug-screening or drug validation purposes (8,9). Because the
We will demonstrate these issues using the GPCR rhodopsin as an line widths of methyl signals in these experiments are relatively
example. Rhodopsin is a glycosylated and palmitoylated 43 kDa pro- narrow as a result of methyl three-site jump and the TROSY selec-
tein containing 348 amino acids. 1H,15N-HSQC spectra of either tion can further increase sensitivity (40–42), observing the methyl
15
N-lysine-labeled or 15N-tryptophan-labeled rhodopsin are shown in signals becomes advantageous for large macromolecular systems,
Figure 2A and B, respectively. The protein was dissolved in 20 mM including membrane protein systems. Several excellent review arti-
sodium phosphate (pH 6.0) and 10% D2O containing octyl glucoside cles describe these techniques (43–46).
or dodecyl maltoside detergent micelles. The quality of both NMR
spectra is quite poor as evidenced by the heterogeneity in number Despite such difficulties in protein expression and sample prepara-
and intensity of signals (Figure 2). Site-directed mutagenesis and tion, there is increasing success in the determination of membrane
screening of solvent conditions has led to the improvement in spec- protein structures by NMR spectroscopy. To illustrate this progress,
tral quality for some membrane proteins, e.g., diacylglycerol kinase, we downloaded a list of membrane protein structures determined
where the E. coli origin and expression system made such studies with the help of NMR spectroscopya and analyzed the structures
possible (38). When an optimal condition for NMR study is not with respect to their transmembrane organization (Figure 3). Of 44
found for the membrane protein of interest, fragments of the pro- structures, 28 structures were determined using solution NMR (the
teins may be studied instead (39). Although such fragments studies others utilized solid-state NMR). While these numbers are encour-
will gain some limited insight into the structure of the membrane aging, it is important to realize that the majority of these structures

A B

Figure 2: 1H,15N-heteronuclear single quantum correlation (HSQC) spectrum of rhodopsin labeled with (A) a-15N-lysine and (B) a,e-15N-tryp-
tophan. Rhodopsin contains 11 lysine residues but only one of these, Lys339, gives rise to a high intensity peak (labeled in the figure) (35).
There are a total of five tryptophan residues in rhodopsin, the signals corresponding to backbone and side-chain signals are represented by
'a' and 'e', respectively (36). Reprinted with permission from the Proceedings of the National Academy of the United States of America (Copy-
right ª 2002, The National Academy of Sciences, Copyright ª 2004, The National Academy of Sciences).


Yanamala et al.

applicability of NMR spectroscopy to the study of proteins in gen-
eral, including protein-ligand interactions. The natural abundance of
these isotopes in the detergents and solvents used can significantly
add to the background, in particular for 1H NMR spectroscopy,
where the 1H isotope is 100% abundant. Additional problems are
the low signal-to-noise ratio because of slow molecular tumbling of
the protein–detergent complex discussed earlier. In the following
paragraphs, we summarize current efforts in overcoming these con-
straints, with major emphasis on recording 1H NMR spectra. Similar
considerations however would also apply to the direct detection of
Figure 3: Analysis of integral membrane protein structures other isotopes such as 13C.
determined by NMR spectroscopy deposited in the protein data-
bankb. The y-axis represents the number of protein structures with Suppression of background signals in NMR
a particular transmembrane segment organization plotted on the x- experiments for membrane proteins
axis. The x-axis represents the total number of transmembrane heli- As described in 'Challenges in membrane protein NMR spectros-
ces in each structure. The '0' category corresponds to b-barrel copy', in the case of membrane proteins, a membrane mimetic is
transmembrane proteins. The PDB identifiers that represent each required, provided by detergent micelles when they are studied with
category are '0' (1G90, 2JMM, 2K0L, 1MM4, 1MM5, 1Q9F, 2JQY, solution NMR methods. The detergent concentrations are typically
2K4T, 2JK4), '1' (1AFO, 2RLF, 1ZLL, 2HAC, 2J5D, 2JO1, 1JP3, 2JWA, 100 times higher than the protein concentrations to ensure that only
2KIK, 2K1L, 2K21, 2K9J), '2' (1WAZ, 2A9H, 2JX4, 2K9P), '3' (2KDC) one functional protein or protein complex is present per micelle for
and '4' (2K73, 2K74). The data for the plot were downloaded on uniformity purposes. The high signal intensity originating from the
November 26, 2009 from Dror Warschawski's websitea. detergent leads to the suppression of signal intensities from the pro-
tein (dynamic range problem) and also results in overlapping with
that of protein peaks. Over-sampling is a feature available in most
still represents either b-barrel proteins (Figure 3, '0¢ bin) or single recent commercial NMR instruments, but if it is not available, large
transmembrane helices (Figure 3, '1' bin). A recent success was the detergent signals also cause other artifacts such as baseline rolling
structure determination of diacylglycerol kinase (Figure 3, '3' bin), and insufficient digitization of the signal (48). An example is shown
which although only consisting of three transmembrane helices for a 0.7 mM solution of rhodopsin in 1% octyl glucoside (Figure 4).
forms a trimer. The trimeric organization is significant because it is At the scale used, the protein signals are not even visible in this
formed via domain-swapping of helices. Thus, the structure actually Figure, and the spectrum is dominated by the detergent signals. A
represents with 9 (!) transmembrane helices the largest membrane value of 1% for the detergent concentration is in fact relatively low;
protein whose structure has been determined by NMR spectroscopy in many cases, much higher detergent concentrations are used,
to date (3). These results are highly encouraging: a decade ago, making the dynamic range problem even more severe.
only the structures of small membrane proteins with molecular
weights less than 10 kDa could be determined by NMR because of A biochemical solution to the detergent background problem is the
the decrease in the molecular tumbling by the addition of deter- use of deuterated detergents. However, their synthesis is typically
gents (47). However, recent developments of NMR methodology and
efforts of protein expression and sample preparation enabled the
earlier mentioned structure determinations for membrane proteins
with molecular weight >20 kDa.

Finally, it should be noted that in the application of NMR-screening
methods to membrane proteins by looking at ligand signals, it is
important to distinguish whether signal changes are because of
ligand–detergent interaction or ligand–protein interaction. It is thus
critical to record a suitable reference spectrum in each case.

1
H NMR-based approaches for membrane
proteins

Solution NMR spectroscopy has dramatically advanced in the scope
of its applicability to proteins, especially when studying proteins of Figure 4: One-dimensional 1H NMR spectrum of bovine rhodop-
increasingly larger size or membrane proteins, by way of using sin acquired in 20 mM sodium phosphate buffer (pH 6.0) and 1%
NMR-active isotopes of hydrogen, carbon and nitrogen. While 1H is octyl glucoside. The spectrum was acquired using a 800 MHz Bru-
100% abundant, 15N and 13C isotopes are used to replace the more ker spectrometer, at 20°C. At the scale shown, only the detergent
abundant 14N and 12C isotopes in proteins, respectively. The ability signals are visible, demonstrating the large difference between the
to introduce these isotopes is therefore one constraint on the intensity of detergent and protein signals.


NMR-Based Screening

very expensive. Unless the protein can be studied in commonly used applicability to membrane proteins, solvent suppression schemes
detergents for which deuterated forms can be purchased off the sometimes with loss of information in some regions of the spec-
shelf, custom-synthesis is often required. In addition, use of deuter- trum are particularly important. The earlier described AIDA method
ated detergent for screening large numbers of samples may (53) also makes use of focusing on a particular spectral region (see
increase the screening cost significantly. The type of detergent that Figure 1). Here, we demonstrate the utility of such an approach
will give rise to optimal NMR spectra while maintaining the func- using selective excitation sculpting studies of full-length rhodopsin
tion of the protein is largely empirical, requiring extensive screening in octyl glucoside micelles as a model system. Rhodopsin is the
of different detergents and detergent ⁄ lipid mixtures and may settle most extensively studied G-protein-coupled receptor, and knowledge
on non-standard detergents (37,49,50). Membrane proteins have to about its structure serves as a template for other related receptors.
be continuously maintained in the presence of membrane mimetics Because of the large numbers of members of the GPCR family and
during cell extraction (or after refolding from inclusion bodies). their importance as drug targets (see Introduction text of this article
Further, all purification and concentration steps require large under Abstract), these studies are highly relevant for drug discovery
volumes of buffers. Because of these reasons, typically the protein efforts involving these receptors.
will be purified in a non-deuterated detergent, followed by
exchange with the deuterated detergent. This adds an additional One-dimensional 1H NMR spectra recorded by selectively exciting
step of complexity to the NMR sample preparation to ensure effi- the protein NH region by applying a selective excitation pulse cen-
cient, homogenous and complete replacement of detergent with tered around 10–12 ppm show 1H chemical shifts from both back-
minimal protein loss. Thus, use of deuterated detergent may not bone and side-chain regions of rhodopsin in octyl glucoside micelles
always be practical based on cost and preparative effort, especially (Figure 5A). Further, excitation of the same region using the hyper-
at the relatively large quantities needed for NMR-based screening. bolic secant shaped pulse to remove detergent and water signals
significantly increased the intensities of the NH peaks in the range
When deuterated detergent is not available, too expensive or not from 6.0–8.5 ppm (Figure 5B) (58,59). Note, however, that the num-
practical, application of multiple solvent suppression experiments, ber of peaks observed in the 1D 1H NMR spectrum is significantly
such as WET (51), selective pulse experiments including sculpting reduced. We tentatively propose that the observed signals arise
(52,53) or coherence selection (54–56), is required. If possible, satu- mostly from the backbone C-terminus residues and flexible loop
ration by radio-frequency is not applied to suppress the water, sol- regions. This hypothesis is based on the previous observation (35)
vent or detergent signals in protein samples because protein that sharp, highly intense and thus slowly relaxing signals are
signals underneath the solvents are also saturated and the signal found only for Lys339 in a uniformly 15N-lysine labeled rhodopsin
reduction is propagated to the entire protein by the spin-diffusion sample (Figure 2). Furthermore, comparison between the observed
mechanism (57). Among the water suppression techniques, pulse signals and those obtained with a peptide corresponding to the
techniques that use relatively long durations are not efficient to be sequence of the C-terminal residues reveals extensive similarities
incorporated into various 3D NMR experiments and coherence between the rhodopsin C-terminus and the free peptide in solution
selection in combination with pulsed-field gradient is commonly (60).
applied.
One-dimensional 1H NMR spectra of bovine rhodopsin recorded at
Because one-dimensional NMR-spectroscopic approaches currently different concentrations of octyl glucoside indicated chemical shift
have (and in the foreseeable future will continue to have) broader dependence of the C-terminus backbone peaks (data not shown),

A B

Figure 5: One-dimensional NMR spectra of unlabeled bovine rhodopsin in octyl glucoside micelles. (A) Selective excitation of the NH
region by employing a selective excitation pulse. (B) Selective excitation of the NH proton peaks with sculpting using hyperbolic secant
shaped pulse (58,59). A total of 0.5 mM (7 mg in 350 lL) concentration of bovine rhodopsin was used to acquire the spectra. The NMR exper-
imental parameters pulse width, excitation bandwidth and acquisition time are as provided in the legend to Figure 7 and detailed in (61).


Yanamala et al.

highlighting the need to control the detergent environment quantita- both the structure and the dynamics of rhodopsin in two different
tively to obtain reproducible NMR results. To investigate possible states, the inactive dark state and the light-activated Metarhodop-
detergent–protein interactions, we recorded one-dimensional and sin II state. The approach is extendable to other conformations,
two-dimensional 1H-1H selective excitation NOE spectra. We such as G-protein-bound or opsin structures.
observed differential interactions of the rhodopsin backbone signals
with those of the detergent micelles (Figure 6). In particular, a set The results obtained with rhodopsin show high promise for the
of strong NOE peaks was observed from rhodopsin protons extension of the approach to other GPCRs. We have already demon-
(Figure 6B, represented by arrows) to a detergent peak at 1.85 strated with rhodopsin that multiple conformations can be studied,
ppm (Figure 6A, indicated by arrow). The identity of this detergent because the life-time of these conformations under the NMR condi-
signal is shown as an inset in Figure 6A, a -CH2- group near the tions studied are known. For other GPCRs, it also needs to be
sugar head group. We did not observe intramolecular rhodopsin established what the stability of resting, activated or G-protein-
protein NOE peaks. A potential solution to detect such NOEs could bound states are, to ensure that the time it takes to acquire an
be provided by detergent deuteration. NMR spectrum is meaningful for the particular conformation of
interest. Furthermore, while the cytoplasmic loops and the C-termi-
Using the sculpting experiments, we have successfully identified nus of rhodopsin are functionally important regions in the protein
novel ligands binding to rhodopsin and interacting with cytoplasmic (critical for receptor activation and G-protein binding), it remains to
loop and C-terminal residues by measuring chemical shift and line- be shown whether the same approach is also suitable to study
broadening effects in selectively excited 1H spectra as a function of ligands such as retinal that bind in the transmembrane domain of
added ligand, the anthocyanin cyanidin-3-glucoside (61). In this rhodopsin.
study, we were able to identify chemical shift and intensity changes
in receptor and ligand. In dark-adapted rhodopsin an upfield shift of
the chemical signals (Figure 7, peaks at position 3, 4, 7, 8, 9 and Saturation transfer-difference (STD) NMR
10) of the protein was observed. In the case of ligand, some of the application to membrane proteins
peaks corresponding to ligand (compare signals at position 2, 11, Of the many techniques developed for screening by NMR, summa-
14, 18 and 19 in Figure 7A with 7D) experienced decrease in inten- rized in 'NMR-based approaches to drug screening', a particularly
sity and some of them disappeared (peaks marked as 'x' and at promising technique for application to membrane proteins is STD.
positions 22, and 24 in Figure 7) in the presence of rhodopsin, indi- The technique requires very small amounts of protein (in the nM–
cating restriction in mobility upon binding. Further, the comparison lM range) because the ligand is present in 100-fold excess over
of the 1H NMR spectra of rhodopsin upon light activation both in the protein (7). Protein signals are saturated by irradiation around
the absence and presence of ligand indicated decrease in peak )1 ppm, which is transferred within 0.1 seconds to the rest of
intensities at peak positions represented as '+' in Figure 7C. Using the protein and the ligand. When the ligand off-rate is fast, the
the selective excitation sculpting method, this study suggested that information is quickly transferred to the ligand in solution where it
the binding of anthocyanin ligand, cyanidin-3-glucoside, modulates decays slowly (within 1 seconds), so that during saturation, the

A

B
Figure 6: (A). One-dimensional
solution selective NOE 1H NMR
spectrum of bovine rhodopsin in
0.15% octyl glucoside recorded at
600 MHz, 25°C. (B). Two-dimen-
sional solution 1H – 1H NOE spec-
trum of bovine rhodopsin in 1%
octyl glucoside. The NOEs from one
of the detergent peaks (marked with
an arrow in Figure 6A) to the 1H
peaks from rhodopsin (represented
in box in Figure 6A) are indicated by
arrows in 6B.


NMR-Based Screening

A

Figure 7: 1H NMR spectra acq-
uired using selective excitation sc-
heme with sculpting. (A) Rhodopsin B
before (black solid line) and after
the addition of ligand, cyanidin-3-
glucoside (red dotted line). (B)
Rhodopsin in the presence of
cyanidin-3-glucoside before (black
solid line) and after light activation
(red dotted line). (C) Illuminated
rhodopsin in the absence (black
solid line) and presence of cyani-
din-3-glucoside (red dotted line).
(D) Cyanidin-3-glucoside alone in C
phosphate buffer and 0.6% dodecyl
maltoside. Each spectrum was
obtained after applying two 180°
hyperbolic secant pulses, following
a 90° rectangular pulse, with
carrier frequency at 11.5 ppm. The
first and second 180° pulses were
employed to invert 6000 and 8000
Hz spectral ranges, respectively.
The last rectangular pulse was
D
applied for 9.9 ls. Echo delay for
the first and the second 180°
pulses were set to be 0.2 and
1 ms, respectively. A total of 2048
scans were acquired with
0.5 seconds repetition delay using
a 800 MHz proton resonance fre-
quency. Reprinted with permission
from the Blackwell Publishing.

proportion of saturated ligands in solution increases, amplifying the direct contact with the receptor to a phenyl ring in the peptide.
difference signal, up until the ligand excess concentration is Only 0.25 nmol of the integrin was sufficient per assay. Another
reached. Thus, the intensity of the STD spectrum will be higher for spectacular application of STD to membrane proteins is the recent
ligands with fast off-rates, but even tight binding can still be mea- study of the interaction of the sweet brazzein protein with the
sured, giving the technique a wide dynamic range. This approach human sweet receptor (62). This receptor is a Class C GPCR, con-
has already been used for study of ligands targeting membrane pro- taining a large extracellular ligand-binding domain, coupled to the
teins by NMR (18,62). In one study, integrins were embedded in seven-transmembrane helical bundle typical for GPCRs. These recep-
DMPC ⁄ DMPG liposomes and binding of cyclic peptides was tested tors are challenging and interesting because they contain multiple
(18). An affinity of 30–60 lM was obtained, typical for this class of binding sites in both transmembrane and extracellular domains and
membrane receptors and demonstrating the particular utility of have very low affinity for their ligands, ranging from lM to mM.
NMR-based approaches to reliably detect relatively low affinities. The ligands can bind simultaneously and affect each other's affinity,
From differences in STD responses of individual protons in the cyc- thus it is imperative that the full-length native receptor is studied.
lic peptide, it was even possible to map the epitope that is in One-dimensional 1H,15N HSQC STD experiments demonstrated the


Yanamala et al.

binding of brazzein to the sweet receptor (100 lg) in membrane determining the structural changes of a protein on ligand binding.
suspensions with high intensity, while a non-sweet mutant brazzein These are probed by changes in line shape and ⁄ or chemical shift
protein did not give rise to strong STD signals. This level of protein of a free fluorinated ligand on binding to a protein (19F ligand-
amounts without purification requirement (because membrane prep- observe studies) or that of a fluorinated residue in a protein on
arations were used) is in our experience relatively straight-forward ligand binding (19F protein observe studies). Both approaches can
to obtain for many GPCRs. Thus, the approach is likely to have be employed in the context of drug screening (19F NMR-based
broad applicability to other membrane receptors. Given that the ligand screening).
STD technique is highly sensitive and neither limited by protein size
19
nor requires the assignment of the protein, this technique should F ligand-observe studies
find wide applicability to screening of ligands for membrane pro- Spectral changes of a free fluorinated ligand on binding to a pro-
teins that have lipid or detergent environments surrounding them. tein – like in the case of 1H NMR – can be either broadening of
its line width or changes in its chemical shift depending on the
binding affinity of the ligand. Fluorine signals of the ligand bound
19
F NMR-based approaches to the protein are expected to show restricted motion compared to
its free state and hence give a broader line shape. It may also
19
F NMR spectroscopy can be a viable alternative for one-dimen- undergo chemical shift changes upon binding that may be either
sional NMR-spectroscopic measurements, providing complementary upfield or downfield depending on the nature of the change of
results. Because there is no background from 19F nuclei in neither interactions of the fluorine atom with its environment. A downfield
biomolecules such as proteins nor detergents used to dissolve shift indicates a more hydrophobic environment or a greater extent
membrane proteins, the applicability range of 19F NMR to study of Van-der-Waals interaction of the fluorine atom. Changes in elec-
ligand binding in soluble and in membrane proteins is identical. In trostatic interactions of the fluorine atom with its environment can
the following paragraphs, we therefore review the extensive litera- influence either a downfield or an upfield shift (63). Note however,
ture on 19F NMR-based approaches to study ligand binding to pro- structural information of the binding site can only be procured by
teins, regardless of the proteins under investigation being soluble observing changes in fluorinated protein on ligand addition.
or membrane proteins. First, we will review 19F ligand-observe stud-
ies using fluorinated ligands, including fluorinated phospholipids. Ligands with a low binding affinity rapidly exchange between bound
We will then cover studies of structure and dynamics of proteins by and free forms that may lead to broadening of its resonances. The
19
F NMR. These studies will involve not only ligand-induced advantage of characterizing ligand–protein interactions of such
changes in structure and ⁄ or dynamics but also those involving other weak binding ligands by studying changes in fluorinated ligands
conformational changes, such as during protein function or protein rather than protein observed changes is the requirement of less
folding, because the principles are the same. amount of protein. Binding constants can be determined by T2 mea-
surements that contain a weighted average of relaxation rates of
19
F NMR studies of protein structure, dynamics and ligand binding the free and bound forms of a ligand at different concentrations
offer several advantages over other NMR-spectroscopic approaches (64). The utility of T2 measurements has for example been demon-
as a result of the unique chemistry of the 19F atom. 19F has 100% strated for BSA in binding studies of isoflurane, a volatile anes-
natural abundance, and its sensitivity to NMR detection is 83% that thetic (64). A Kd of 1.4 mM was obtained from T2 measurements of
of 1H. The presence of nine electrons surrounding the 19F nucleus the free ligand and that bound to the protein (64). Another interest-
makes it very sensitive to minor changes in its environment, including case is the influenza virus M2 membrane protein, which forms
ing both Van-der-Waals and electrostatic interactions, which is proton channels that lead to the disruption of the matrix protein
reflected in its wide range of chemical shifts. This characteristic and the release of the viral genome (65). Amantidine is an inhibitor
increases the probability of obtaining well-resolved peaks of fluo- of this process. 1H NMR of amantidine or the protein could not pro-
rine atoms in different environments. Another major advantage of vide information on ligand binding because very broad signals were
19
F NMR over other conventional NMR techniques is the appear- obtained (66,67). 19F T2 relaxation measurements were used in this
ance of its NMR signals in the absence of any background signals, case to reveal interactions between the fluorinated amantidine
including membrane mimetic environments and even entire cells. ligand, and the M2 protein as well as interactions between the
The information content of 19F NMR ligand-based screening, while ligand and the dodecylphosphocholine micelles the protein was
not as high as SAR by NMR, is higher than that of HTS methods, dissolved in (67).
in particular those employing cell-based approaches. These unique
properties of the 19F nucleus suggest that 19F NMR spectroscopy Inhibitors of enzymatic reactions may be detected by a method
could provide a highly desirable alternative to HTS by conventional called fluorine-based biochemical screening (FABS) (68,69). In this
NMR-spectroscopic techniques, in cases where the latter methods method, a substrate is tagged with a fluorinated moiety, and
may not be applicable, such as for many membrane proteins or for changes in distinct 19F signals for the substrate and product are fol-
in-cell studies. From a practical perspective, 19F labeled compounds lowed with the progress of an enzymatic reaction in presence of
are easily accessible by different chemical methods (see 'Synthesis test inhibitors. This method is particularly suited for screening inhib-
of 19F containing small molecule compounds'). itors with low-binding affinity that remain undetected by regular
NMR ligand screening methods. The sensitivity of the method is
Ligand–protein interaction studies include (i) evaluating binding of enhanced in the case of weak affinity ligands by having moieties
ligands, (ii) characterizing binding kinetics of the ligands and (iii) with three fluorine atoms attached to the ligand and the method is


NMR-Based Screening

named 3-FABS (69). IC50 value of the inhibitors is obtained by tak- activated cysteines. This approach has been shown to work well
ing the ratio of the integrals of the 19F peaks of the substrate and with GPCRs (73). However, this method is limited to labeling only
the product as a function of inhibitor concentration. In addition to surface exposed amino acids or those amino acids in membrane
screening mixtures of inhibitors, it is also possible to screen mix- proteins for which side chains are exposed to the membrane for
tures of closely related enzymes to determine selectivity of an ease of entry of labeling reagents. The principle is shown in
inhibitor provided the substrate is specific for the different enzymes. Figure 8. A receptor will have endogenous cysteines, shown in a
This method has been applied in several cases such as screening homology model of the corticotropin-releasing factor receptor in
inhibitors for kinase AKT1 and protease trypsin (69), caspases (70) Figure 8A. The cysteines can be derivatized with a 19F containing
and thymidine phosphorylase (71). ligand directly, but a less invasive approach is to first activate the
accessible cysteines and then introduce a trifluoroethylthiol group
Information on binding constants and stoichiometry of binding can through disulfide exchange (Figure 8B). This procedure contains min-
be obtained by titrating fluorinated ligand and monitoring the imal perturbation from added chemical groups and retains maximal
changes in the protein-bound peaks and free peaks of the ligand by flexibility from the ethyl side chain.
19
F NMR. In the slow exchange regime, we will observe two peaks,
which may be sufficiently resolved in their chemical shift values to Using 19F NMR to observe the protein can be useful, for example,
be useful for quantitation. Binding constants are determined from if it is of interest to determine whether a receptor is in an active
the ratios of bound and free ligand concentrations quantified by or inactive conformation upon ligand binding. If the specific chemi-
integrating 19F NMR signals (72). cal shifts associated with each state are known, then the appear-
ance of the respective peaks can be used as an indicator whether
a ligand is, for example, an agonist or antagonist or an inhibitor or
19
F protein observe studies inducer of oligomerization. This idea is illustrated with bovine rho-
Studying ligand binding by monitoring changes in 19F signals report- dopsin: 19F NMR spectroscopy was used to study the conforma-
ing on protein conformation can be useful under conditions where tional changes in rhodopsin upon light activation to which the 19F
accurate affinities and binding modes cannot be unambiguously chemical shifts were very sensitive (73). In this case, the 19F label
determined from ligand-observe methods, or where it is desirable was introduced through chemical reaction of trifluoroethyltiol with
to increase the information content that can be obtained from 19F activated cysteines (Figure 8B), here on rhodopsin. Distinct chemical
NMR studies. If 19F labels are placed on the protein, one can study shifts are found for the dark, inactive and the light-active states at
where the ligand binds, and whether the ligand induces conforma- numerous sites on the rhodopsin surface (Figure 9).
tional changes, oligomerization or folding transitions. There are two
approaches to introduce 19F labels into proteins. In the first Determining structural changes in specific regions of a protein on
approach, a 19F label is introduced biosynthetically as a fluorinated ligand binding requires the introduction of a 19F label into the pro-
amino acid. As for incorporation of other isotope-labeled amino tein. More common than the chemical cysteine-labeling approach,
acids (see above), this method may not be very cost effective for is to substitute amino acids in the protein with fluorinated analogs
mammalian membrane proteins (including GPCRs) because insect or and track the chemical shifts and line widths in 19F NMR spectra.
mammalian cell expression required for such systems in fluorinated The small size of the fluorine atom has enabled the substitution of
amino acid-rich medium can be very expensive. In the second residues such as Trp, Tyr, Phe with their fluorinated analogs without
approach, a 19F label is introduced through chemical reaction with perturbations of the native structures of proteins. The observed

A B

Figure 8: Selective cysteine
CF3-derivatization of G-protein-
coupled receptors (GPCRs). (A) As
an example, the five endogenous
cysteine residues in the corticotro-
pin-releasing factor receptor (CRFR)
are shown in yellow. (B) Chemical
procedure of selective cysteine
derivatization via activation and
thiol exchange (73,74). A sulfhydryl
group on the protein (here GPCR)
is activated by reaction with
dithiodipyridine. The thiopyridinyl
derivative undergoes disulfide
exchange with a fluorine-contain-
ing sulfhydryl reagent (73).


Yanamala et al.

I 4-fluoro isomer labeled protein can be produced in much larger
quantity and shows no perturbations of the native structure (75).
Assignments of the 19F peaks can be performed by either mutating
the fluorinated residue or by nudge mutations, whereby a mutation
in an adjacent position changes the chemical shift of the
fluorinated residue as a result of change in its environment, or by
complexation of a solvent accessible fluorinated residue with
II
paramagnetic ions such as Gd3+ leading to line broadening of that
residue (76).

19
F NMR has been used to track both allosteric and non-allosteric
changes on ligand binding to a protein. For example, in studies of
the binding of D-glucose and D-galactose to the fluoro-tryptophan-
III labeled aqueous chemosensory receptor of E. coli (77), it was seen
that sugar binding resulted in changes in chemical shifts of not only
those fluoro-tryptophan residues that are adjacent to the binding
site but also those tryptophan residues that are distant from the
bound sugar by as much as 15 Š (77). These results indicate that
sugar binding leads to a global change in the structure of the pro-
IV tein that is translated from the binding site to distant regions on
the surface, and this global change can be tracked by 19F NMR
(77). A different way of probing conformational change is to
observe line broadening by the addition of Gd(III)-EDTA that indi-
cates solvent accessibility of the fluorine-labeled residue (78). Infor-
mation on binding constants and stoichiometry can be obtained by
V titrating the ligand and monitoring the shifts in the peaks of fluori-
nated amino acids (78). 19F NMR has also proved to be suitable for
studying protein dynamics by monitoring relaxation rates of fluori-
nated residues, as illustrated by the study of ligand binding in iono-
tropic glutamate receptor (GluR2) (76).

VI Structure and function of membrane proteins in particular are lar-
gely influenced by their interactions with lipid bilayers, and 19F
NMR can be used to study the detailed mechanisms of these
effects. For example, line widths of lactate dehydrogenase become
sharper on adding increasing concentrations of lysolecithin in a
non-linear fashion (75). Because there was no change in the chemi-
cal shifts of the tryptophan residues, it was concluded that lysoleci-
Figure 9: 19F one-dimensional NMR spectra of trifluoroethylthi- thin is only solvating the protein and not causing a conformational
ol-labeled bovine rhodopsin, and its various cysteine mutants in change (75). The number of lipid molecules bound to a protein can
dark (red lines) and after illumination (blue lines) (73). The spectrum be calculated from the variation in line width with lipid concentra-
was referenced with respect to trifluoroacetic acid (TFA). Reprinted tion. In the case of lysolecithin binding to lactate dehydrogenase,
with permission from the Proceedings of the National Academy of this number was found to be lower than the aggregation number of
the United States of America (Copyright ª 1999, The National lysolecithin, suggesting that lactate dehydrogenase is not inserted
Academy of Sciences). in the micelles but binds individual lipid molecules that shield
exposed hydrophobic surface patches from initiating aggregation
and inactivation of the enzyme (75).
chemical shift range, expression of the labeled protein in sufficient
19
amount and integrity of the fluorine labeled protein are some of F NMR is a suitable technique in mapping the sites of the inter-
the factors that should be considered when choosing an isomer of action of proteins with membranes. The use of solvent induced iso-
a fluorinated amino acid. For example, of the 4-fluoro, 5-fluoro and tope shifts can provide information on solvent exposure of a
6-fluoro isomers available for fluoro-tryptophan, the 6-fluoro isomer residue. However, residues that are not solvent exposed could be
has a very narrow chemical shift range and also shows broad either buried in a protein core or face the membrane or be mem-
unresolved spectra compared to the other two fluoro-tryptophans in brane bound. This ambiguity can be overcome by the use of fatty
lactate dehydrogenase enzyme (75). Moreover, the 6-fluoro isomer- acids in which a paramagnetic spin label is incorporated into the
labeled protein shows perturbations in its secondary structure as membrane under study, and its interaction with a fluorine probe in
detected by circular dichroism spectroscopy, and a broad peak is the protein is detected by the broadening of the corresponding fluo-
obtained in the 19F NMR spectrum (75). On the other hand, the rine peaks in a 19F NMR spectrum (79). The paramagnetic electron


NMR-Based Screening

of the labeled fatty acid 7 Š from either end of the lipid phase will based competition-binding experiments in which 19F NMR signals of
cause broadening of a fluorine nucleus that is within 15 Š from the a spy molecule, which has medium to weak affinity for the protein
label i.e., either in or near the lipid phase (79,80). By labeling spe- of interest, is monitored as it is displaced by higher affinity ligands
cific amino acids with 19F and by their mutagenesis analysis, inter- during a screen (83,84). This places a constraint on the types of
actions with lipids can be followed, thus helping in mapping sites ligands that can be identified with this method, as the ligands have
of protein–lipid interaction. The amount of broadening observed is to exhibit sufficient affinity to compete with the spy molecule,
inversely proportional to the distance between the label and fluori- thereby limiting the affinity range of binders. Another limitation is
nated residue raised to the power of six (78). 8-doxylpalmitic acid that as in other competition binding experiments, this method can
incorporated in lysophosphatidylcholine is used as the nitroxide only study ligand binding to previously known binding site. Control
spin-labeled fatty acid to map the site of interaction of lactate molecules, which do not interact with the protein, can also be used
dehydrogenase with lysophosphatidylcholine (80). Another use of along with the spy molecule in this method. Therefore, the screens
such spin-labeled fatty acids, in the case of lactate dehydrogenase, are performed by monitoring the relative signal intensities of the
is to determine whether substrate binding has any effect on the spy and the control molecule (83,84). The protein is then added to
residues in the lipid binding region. Lactate dehydrogenase oxidizes the mix of spy and control molecule and the NMR signal of the spy
D-lactate, and the electrons produced reduce the nitroxide labeled molecule disappears as a result of binding to the protein (83,84). A
fatty acid, disrupting its interactions with the fluorine nucleus and hit in the screening process is indicated by the reappearance of the
recovery of the peak that was lost ⁄ broadened because of its inter- spy molecule signal at the same place as before the protein was
action with the label (80,81). added indicating displacement of the spy molecule with a com-
pound of higher affinity from the library (83,84). The extent of dis-
placement can be measured from the ratio of the control to spy
19
F NMR-based ligand screening molecule signal intensity that will in turn provide the binding con-
The ease of obtaining information from ligand-binding studies by stant of the hit (83,84). The choice of the spy and control molecules
19
F NMR, as mentioned earlier, has extended its applicability to can be decided by their solubility in aqueous solution so that non-
HTS of chemical libraries that is a routine procedure in the field of specific binding to proteins can be ruled out. A major advantage of
drug discovery. The broad chemical shift dispersion of the fluorine this method is the requirement of only the spy molecule to be fluo-
nucleus allows for identifying 'hits' in a screen with less chances of rinated and not the ligands being screened. This approach is known
encountering the problem of spectral overlap from different chemi- as fluorine chemical shift anisotropy and exchange for screening
cal compounds. The simplicity of the 19F spectra, unlike 1H spectra, (FAXS) (83). The FAXS method has been successfully used to screen
decreases the time for deconvoluting the spectra when a large mix- libraries for human serum albumin where the binding constant of a
ture of chemicals is being screened. Changes in chemical shift val- hit was found to be in good agreement with that obtained from
ues and ⁄ or line widths of the free fluorinated ligand upon the other techniques such as fluorescence spectroscopy and isothermal
addition of a protein will indicate whether a compound is binding titration calorimetry (85). Human serum albumin concentrations as
to the protein or not. Thus, monitoring free ligand peaks allows the low as 600 nM were used (85), showing that the use of very low
use of very low protein concentrations, in tens of lM range. Infor- protein concentrations is a major advantage of FAXS over other
mation on binding constants and stoichiometry of binding from NMR-screening methods. This is especially beneficial for finding
ligand titration experiments can be further used to rank order potential ligands for membrane proteins that are important drug tar-
ligands in a screen. Such information was obtained while screening gets but are difficult to be purified in large amounts. This method
a library of compounds for chaperones PapD and FimC, involved in was also used to screen ligands for the kinase domain of p21-acti-
the assembly of pili on E. coli, and are essential proteins that vated kinase (84). Apart from its use in HTS, FAXS has been very
represent targets for the development of antibacterial agents (82). suitable for fragment-based screening of potent ligands. The use
19
F NMR studies can also be used to provide further information on has been illustrated in screening fragments against v-Src SH2
binding sites to optimize the lead compound by characterizing the domain that has a high affinity for phosphotyrosine (86).
structural changes induced by their binding. This is performed by
using proteins substituted at different positions by fluorinated For HTS of ligands, ligand titrations to obtain binding affinities are
amino acids and monitoring their chemical shift changes on ligand not always feasible because of (i) time-consuming titration proce-
binding. This is much less expensive and easier compared to dure and performing relaxation experiments for each titration point
1
H NMR where the spectra are complicated and further (ii) aggregation arising from addition of excess ligand during titra-
deconvolution requires expensive isotope labeled samples of high tions for ligands with medium affinities and (iii) loss of native char-
concentration. acteristics of a protein by the addition of the increasing
concentrations of ligands dissolved in organic solvents. A different
There is a concern regarding availability of a library of fluorinated 'titrationless' method has been developed based on gxy and R2
compounds. However, about 12% of the compounds in Available measurements (87). gxy is transverse cross-correlation rate constant
Chemical Directory of Screeningd compounds contain fluorine. As of a fluorine attached to an aromatic ring and its ortho-proton and
described earlier, there are a few drawbacks of the ligand-based R2 is the transverse relaxation rate constant. The ratio gxy ⁄ R2 gives
screening methods if the ligand (i) has very high affinity because of a more accurate estimation of the exchange rate constant than that
the insensitivity of NMR to detect ligand peaks in sub-lM concen- obtained from the more conventional R1q (rotating frame relaxation
tration ranges (ii) has slow kinetics and (iii) binds to the protein via rate) measurement. This in turn gives a more accurate dissociation
a covalent bond. However, these problems are overcome by ligand- constant of the ligand (87).


Yanamala et al.

As a proof of concept for extending these approaches to membrane Comparison of 1H and 19F-NMR-based
proteins, we screened binding of 19F-labeled small molecules to versus conventional screening of
rhodopsin by mixing the ligands with the receptor. Ligands were in membrane proteins
a mixture of 10 compounds at 50 lM concentration each. The
receptor concentration was 0.2 mM in detergent solution (fivefold The main advantage of drug discovery by NMR spectroscopy when
excess). For a ligand with micromolar affinity, these conditions compared to traditional HTS methods using other spectroscopic or
ensure that the majority of the ligand will be bound, and therefore cell-based assays is its high-information content: in addition to
a maximal peak shift is expected for a hit. Excellent signal-to-noise ligand binding itself, the location of binding, affinities and confor-
ratio can be achieved with 7 min acquisition time (Figure 10). Both mational changes induced in the protein can be observed. Further-
line-width and chemical shift changes were observed. more, as a result of the high sensitivity of NMR spectroscopy to
molecular size, artifacts arising from low solubility of the ligand or
A fragment-based library can be considered complimentary to a ability of the ligand to precipitate the protein virtually never go
library of compounds for HTS purposes. Such a library is a collec- undetected, unlike in traditional HTS approaches. However, the
tion of fluorinated fragments based on Local Environment of Fluo- stringent requirements are also the main disadvantage, limiting the
rine (LEF) (88). The collection of chemical fragments covers a applicability of traditional NMR-based approaches to small soluble
wider chemical space than HTS libraries, and the 'hits' obtained proteins. However, these difficulties can be overcome by using spe-
in a fragment library screen would lead to faster 'lead' optimiza- cialized 1H-based approaches and 19F-NMR-based approaches,
tion. Many parameters are kept in mind during the building of which open the door to study of proteins that are otherwise out of
such a fluorinated fragment library. For example, local substituents reach for NMR, including large and ⁄ or multimeric soluble protein
around the fluorine atom influence its chemical shift dispersion complexes and full-length membrane proteins in detergent micelles.
and solubility. Usually, a single chemically equivalent fluorine is
preferable, because more than one non-equivalent fluorine atom An example demonstrating the limitations in traditional HTS meth-
would lead to complex 19F NMR spectra. The fragments are clus- ods is the most common membrane protein drug discovery target
tered according to their global structural features and local envi- family the GPCRs. Because GPCRs are not enzymes and have tradi-
ronmental fingerprints into different global and local clusters so tionally in the past been difficult to obtain in soluble form, all cur-
that the library has a good coverage of different environments rent HTS assays are cell-based. Several different approaches are
around the fluorine atom. These fragments are then mixed into typically employed. Changes in intracellular calcium concentration
two batches: one for CF3 containing molecules and the other for are measured for Gq coupled receptors, the cAMP assay is used for
CF-containing molecules. The fragments are screened by collecting Gi or Gs coupled receptors. More recently, reporter genes have
19
F NMR spectra in the absence and presence of a protein and been employed, beta-arrestin redistribution has been measured, and
considering those signals as 'hits' that are perturbed on protein receptor internalization has also been used as a reporter for GPCR
addition. The screening can be further confirmed by recording the ligand binding and activity (89). The most sensitive and widely
same spectra in the presence of a known ligand. The advantage employed assay is the cAMP assay, but it is restricted to Gs and Gi
of this method is that it uses fewer concentrations of the frag- coupled receptors. The calcium-based assay employed for Gq cou-
ments, thus enabling the testing of a large compound mixture and pled receptors has the problem of not distinguishing constitutive
also lowering the protein concentration to be used. The low frag- activity from basal levels of intracellular calcium concentration, it is
ment concentration is also helpful in not limiting the use of being difficult to quantitate pharmacological effects. The reporter
ligands that have low water solubility. gene assay requires long incubation with ligands, and there are

Figure 10: Example of a screening of a 19F-labeled compound library (eight compounds are visible in the particular range shown). The 19F
NMR spectra were acquired both in the absence (colored blue) and presence of bovine rhodopsin (colored red). The buffer used to acquire
both the spectrum contained 50 mM phosphate buffer (pH 6.0) and 0.5% dodecyl maltoside micelles.


NMR-Based Screening

many problems associated with this, including many false positives, mostly deadly condition. This is an interesting example because the
issues with stability, redistribution of ligands and receptors, etc. compound is by no means 'drug-like', containing three strong elec-
Arrestin redistribution is a protein interactions-based assay: arrestin trophiles in addition to a nitro group. Nevertheless the compound is
binding to the GPCR is initiated by the phosphorylation of the C-ter- well tolerated, no severe side effects are reported and the drug
minus of the GPCR. It has been demonstrated in many instances comprises a major therapeutic advancement by increasing the for-
that binding of proteins at the cellular side of the receptor, includ- mer 4-year survival rate of 29% of newborns with HT-1 to 88%.
ing arrestin binding to the C-terminus, but also other proteins, e.g., Because nitisinone is probably an exception rather than the rule
those involving PDZ domains, alters the ligand-binding properties and many of the fluorinated compounds that are commercially avail-
and pharmacology of receptors. Finally, receptor internalization is a able will not have the desired properties to make a drug or even
complicated process, and efficient and fast internalization is not screen for biologically relevant compounds, the development of new
always given. In addition to these assay-specific disadvantages, all libraries containing 19F is highly desirable. Introduction of fluorine
of these assays are necessarily indirect and are therefore error- in organic compounds is an established area of organic chemistry
prone. Moreover, compound libraries have limited solubility, and and can be accomplished by a plethora of techniques (92). Many
high concentrations of DMSO are needed to solubilize them. These useful reactions exist to selectively introduce fluorine in organic
high DMSO concentrations alter the cell surface properties. Finally, compounds (Figure 11). To this end specific reagents have been
while an HTS will almost always yield a hit, especially when introduced, e.g., the recently described Togni's reagent for the elec-
screening large libraries, the quality of the compounds identified trophilic introduction of trifluormethyl groups (93) or Buchwald's
may be low and time-extensive and cost-extensive procedures are nucleophilic aromatic substitution of triflates (94) (Table 1).
required to transform the hit to a lead.
Because of the exceptional physico-chemical nature of fluorine,
NMR-based screening has found increasing application to soluble however, organic chemistry of fluorine often takes different reaction
proteins not only because of the enormous amount of information pathways (Table 1). Thus, fluorine introduction is commonly used in
that can be obtained from such a screen (12,90), but most impor- medicinal chemistry to alter the drug compound's profile, including
tantly, NMR-based assays are not prone to artifacts brought about by its solubility, metabolism, pKa and logD (lipophilicity). In addition, it
denaturation, aggregation or precipitation of proteins induced by the is well known that there are distinct stereochemical effects in fluo-
ligands. There are estimates that 20% of all hits in HTS are based on rine compounds as opposed to their non-fluorine counterparts, e.g.,
unspecific ligand effects. Such effects are immediately recognized in the trifluormethyl group in phenols has an energetic preference for
NMR-based screens because of the direct measurement of protein an out-of-plane geometry as opposed to the methyl group (Table 1,
signals. Furthermore, solubility of the compounds is directly visible
from the NMR samples. Another advantage is the fact that weak
ligands can be identified easily. A weak but selective ligand can
become the starting point for successful screening, such as is
exploited in the fragment-based screening approach. Thus, even
though an NMR-based screen may seem more expensive because of
the large protein requirements, in the long run, successful compounds
may be found easier and cheaper when viewed from the end-product
perspective. Typically, HTS is evaluated based on the number of com-
pounds screened versus number of hits, but one really has to critically
evaluate how many of the hits have actually led to a lead or drug. In
fact, there are many cases where HTS in pharmaceutical industry has
not yielded drugs against a desirable target.

Synthesis of 19F containing small
molecule compounds

The access to diverse and drug-like screening libraries labeled with
19
F is the prerequisite for 19F NMR-based screening technology. A
recent database search revealed that more than million fluori-
nated small molecular weight compounds are commercially avail- Figure 11: Overview of some current fluorine chemistries. A lar-
ablec. However, many of those compounds do not satisfy drug-like gely underdeveloped way to access fluorine-containing organic com-
criteria and are rather unlikely to yield expandable hits during pounds is by using multicomponent reaction chemistry (MCR) and
screening. A notable exception is the trifluoromethyl group contain- employing fluorine building blocks (95). Many fluorinated building
ing compound nitisinone. This compound was originally developed blocks are commercially available in large diversity, e.g., aldehydes,
and is still used as an herbicide. It was recently found to be useful carboxylic acids, amines, alcohols, cyanates, phenols and heterocy-
to treat the hereditary orphan disease tyrosinemia type 1 (HT-1) cles. Based on the scaffold diversity amenable by MCR chemistry
(91). Since its first use for this indication in 1991, it has replaced one can easily imagine the accessible fluorine chemical space
liver transplantation as the first-line treatment for this rare and (Figure 12).


J.1747 0285.2009.00940.x

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