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Week 3- Protein Folding and Structure.pdf

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Week 3- Protein Folding and Structure.pdf

  1. 1. Protein Folding and Structure January 23, 2022
  2. 2. Protein Structure  The arrangement and linking of amino acids to form a functional protein is viewed in a stepwise fashion  Primary structure – linear order of amino acid residues in a protein  Secondary structure – three dimensional form of a protein  Tertiary structure – three dimensional shape of a protein  Quaternary structure – arrangement of multiple protein subunits in a multimeric protein complex
  3. 3. Primary Protein Structure  The linear order of amino acid residues along the polypeptide chain  Amino acids can be abbreviated by 3 letters or single letter  For example: Alanine = ala or A; Lysine = lys or K Example - Chymotrypsin • Enzyme that degrades other proteins • 263 amino acids • 27,713 Da
  4. 4. Primary Protein Structure  Insulin is a small protein that consists of two polypeptide chains that are covalently bonded  The A chain is 21 amino acids long while the B chain is 30 amino acids long  The two polypeptide chains are linked via a –S-S- bond (called cystine)
  5. 5. Secondary Protein Structure  The primary structure leads to the Secondary Structure  The secondary structure refers to the folded structures that form within the polypeptide chain due to interactions between atoms of the backbone  Held in shape by hydrogen bonds and are more or less independent of the R-groups  Most common types of secondary structure are a helix and the b pleated sheet
  6. 6. Secondary Protein Structure  a-Helix structure  The carbonyl (C=O) group of one amino acid is hydrogen bonded to the amino hydrogen (N-H) of an amino acid that is four residues down the chain  This pulls the polypeptide chain into a helical structure that resembles a curled ribbon with each turn of the helix containing 3.6 amino acids  The R-groups of the amino acids stick outward from the a-helix, where they are free to interact  b-Pleated sheet  Two or more segments of a polypeptide chain line up next to each other and form a sheet-like structure held together by hydrogen bonds  The hydrogen bonds form between carbonyl and amino groups of the backbone, while the R-groups extend above and below the plane of the sheet  Strands of the b-pleated sheet may be parallel, or pointing in the same direction (such that the N- and C-terminus match up) or antiparallel, or pointing in the opposite direction (such that the N-terminus of one strand is positioned next to the C-terminus of the other)
  7. 7. Secondary Protein Structure Example of an a-helix and b-pleated sheet in a protein 3.6 residues per turn
  8. 8. Arrangement of Amino Acids in a-Helix Ribbon Diagram of an a-helix Hydrophilic side Hydrophobic side
  9. 9. Parallel vs Antiparallel in b-Sheet Parallel b-Pleated Sheet Antiparallel b-Pleated Sheet
  10. 10. Secondary Protein Structure: Additional Information  Certain amino acids are more or less likely to be found in a-helices or b pleated sheets  Proline is known as a “helix breaker” due its unusual R group that creates a bend in the peptide backbone structure that is not compatible with helix formation  The aromatic amino acids (Trp, Tyr, Phe) are often found in b pleated sheets  Many proteins contain both a-helices or b pleated sheets; some contain just one type while others do not form either
  11. 11. Chymotrypsin Secondary Protein Structure  Bovine alpha-chymotrypsin: an example of a protein that has both a-helices or b pleated sheets a-helix b-pleated sheet
  12. 12. Tertiary Protein Structure  The overall three-dimensional structure of a polypeptide is called its tertiary structure  The tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein  This includes hydrogen bonding, ionic bonding, dipole-dipole interactions, and van der Waals forces  A critical component to tertiary structure are hydrophobic interactions, in which amino acids with nonpolar, hydrophobic R groups cluster together on the inside of the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules  Disulfide bonds can also contribute to tertiary structure  Can be both inter-strand (between two polypeptide strands) or intra-strand (within the same polypeptide)
  13. 13. Tertiary Protein Structure - Interactions  An example of the various interactions that can lead to a proteins tertiary structure Polypeptide backbone Ionic bond Hydrophobic interactions Disulfide linkage Hydrogen bond
  14. 14. Tertiary Protein Structure of Chitinase Structure of the barley chitinase • Chitinase is an enzyme that cleaves chitin, which is a polysaccharide found in fungi, plants and insects • The side chains of the catalytic acids are shown in green; side chains of several residues that are (putatively) involved in substrate binding and catalysis are shown in red and purple Chitin Chitinase
  15. 15. Tertiary Protein Structure of Triose Phosphate Isomerase Triose phosphate isomerase Dihydroxyacetone phosphate D-glyceraldehyde 3-phosphate Active Site Forms a b- barrel
  16. 16. Quaternary Protein Structure  For proteins that have only one single polypeptide chain, the tertiary structure is the most resolved protein structure  However, for proteins that are made up of multiple polypeptide chains (also known as subunits), the combination of all of these subunits is called the quaternary structure  The same types of interactions that contribute to tertiary structure also hold the subunits together to give quaternary structure  An example of a protein with quaternary structure is hemoglobin
  17. 17. Hemoglobin Structure  Hemoglobin is a iron-containing oxygen transport protein found in erythrocytes (red blood cells)  Composed of four polypeptide chains (tetramer), consisting of two a and two b subunits (a2b2)  Each subunit has a MW of 16 kDa for a total MW of 64 kDa  Each subunit contains a tightly associated heme group that is bound to iron  Oxygen binds to the heme component of the tetramer in a cooperative fashion for a total of 4 oxygen molecules per tetramer  As the first oxygen molecule binds, the tetramer’s conformation changes to promote the binding of the remaining three oxygen molecules
  18. 18. Quaternary Protein Structure Structure of human hemoglobin. α and β subunits are in red and blue, respectively, and the iron-containing heme groups in green Hemoglobin heterotetramer – a2b2
  19. 19. Myoglobin Structure  Myoglobin is a heme-containing protein that is found in muscle tissue, where it binds oxygen, and helps provide extra oxygen to release energy to power muscles  Is a monomeric protein with 153 amino acid residues  MW of 16.7 kDa  Contains a tightly associated heme group that is bound to iron • Oxygen binds to the heme component of the protein • Oxidation of iron (Fe+2 to Fe+3) is responsible for the red color of muscle and blood
  20. 20. Hemoglobin Binding to Oxygen is Cooperative % O 2 Saturation PO2 (mm Hg) Hemoglobin (sigmoidal) Myoglobin (hyperbolic) tissues lungs Amount of O2 dissolved in the blood • Hemoglobin is primarily responsible for the transport of oxygen to tissues • Myoglobin is responsible for oxygen storage
  21. 21. Protein Folding  In order for proteins to achieve their tertiary (or quaternary) structure, the protein must form the appropriate conformation – this is called protein folding  Protein folding is a spontaneous process that is primarily guided by hydrophobic interactions (e.g. hydrophobic effect), hydrogen bond, ionic bonds and van der Waals forces  Protein folding must be thermodynamically stable  Chaperones are a class of proteins that aid in the correct folding of other proteins  Chaperones are shown to be critical in the process of protein folding in vivo because they provide the protein with the aid needed to assume its proper alignments and conformations efficiently enough to become "biologically relevant"
  22. 22. Protein Denaturation  When a protein loses its 3-dimensional structure and reverts into an unstructured string of amino acids, this is called protein denaturation  Denatured proteins are usually non-functional  In some cases, denatured proteins can be reversed, sometimes it cannot  Proteins can be denatured when heated or exposed to high salt solutions such as urea (6 M) or guanidine HCl  An example of a denatured protein is egg white (egg albumin); once heated or vigorously stirred, it becomes denatured and will not return to its original state
  23. 23. Protein Denaturation – Egg Whites  Egg whites consist primarily of water and egg albumin; albumin consists of a number of proteins  It can be denatured upon agitation or heat Agitation Folded Protein Unfolded Protein
  24. 24. Protein Structure Determination  There are several methods currently used to determine the structure of a protein; these are:  X-ray crystallography  NMR  Three dimensional electron microscopy (CryoEM)  X-ray free electron lasers (XFEL)
  25. 25. X-Ray Crystallography Overview  X-ray crystallography can provide a detailed “picture” of a proteins structure, including atomic details such as ligands, inhibitors, ions, etc.  A protein must be purified and crystallized, then subjected to an intense beam of X-rays  The protein in the crystal diffracts the X-ray beam into one or another characteristic pattern of spots, which are analyzed to determine the distribution of electrons in the protein  The resulting map of the electron density is then interpreted to determine the location of each atom  Two types of data are collected: The first are coordinate files, which include atomic positions for the final model of the structure; the second are data files which include the structure factors such as the intensity and phase of the X-ray spots in the diffraction pattern
  26. 26. X-Ray Crystallography Process  Workflow consists of three basic steps  Step 1: produce an adequate protein crystal  Step 2: place in an intense beam of X-rays (single or variable wavelength) to produce a regular reflection pattern  Step 3: the collected data is combined with chemical information to obtain and refine a model from the arrangement of atoms – this is called a crystal structure
  27. 27. X-Ray Crystallography Process  Crystallization  Generation of a diffraction-quality crystal is the biggest concern  Need a pure crystal of high regularity  Many methods available to grow crystals, such as gas diffusion, liquid phase diffusion, temperature gradient, vacuum sublimation, convection, etc.  Data Collection  X-ray irradiation causes the crystal to be diffracted, and the diffraction data are recorded  Data Analysis  Two-dimensional diffraction patterns corresponding to a different crystal orientation is converted into a three-dimensional model of the electron density, which is completed by Fourier transform analysis  Initial phasing, model building and phase refinement are the final steps in finalizing a protein structure; in some cases this may require additional studies such as molecular replacement or heavy atom methods
  28. 28. X-Ray Crystallography – Diffraction Pattern  Diffraction pattern of Myoglobin – which is a heme- containing protein which carries and stores oxygen in muscle Myoglobin was the first protein structure solved by X-ray crystallography; this led to a Nobel prize for John Kendrew and Max Perutz
  29. 29. X-Ray Crystallography  Good atomic resolution (e.g. 1 or 2 Angstroms) provides an outstanding picture of the protein, including locations of each atom and how it relates to the protein
  30. 30. X-Ray Crystallography Facility  X-ray crystallography facility consists of a electron/beam source, sample and detector  Sample prep (i.e. crystal formation) can be partially automated with
  31. 31. NMR Spectroscopy  Nuclear Magnetic Resonance (NMR) spectroscopy is another method that can be used to determine the structure of a protein  The protein is purified and place in a strong magnetic field, and then probed with radio waves  A distinctive set of observed resonances may be analyzed to give a list of atomic nuclei that are close to one another, and to characterize the local conformation of atoms that are bonded together  This list of restraints is then used to build a model of the protein that shows the location of each atom  The technique is currently limited to small or medium proteins (<35 kDa), since large proteins present problems with overlapping peaks in the NMR spectra.
  32. 32. NMR Spectroscopy  A major advantage of NMR spectroscopy is that it provides information on proteins in solution, as opposed to those locked in a crystal or bound to a microscope grid – thus, NMR spectroscopy is the premier method for studying the atomic structures of flexible proteins  Analysis is far more complex than with simple small organic molecules  Multidimensional techniques, such as nuclear Overhauser effect (NOE) experiments must be utilized which require labeling the protein with 13C and 15N  NOE experiments measure distances between atoms with the protein; this distances allow generation of a 3-dimensional structure of the protein
  33. 33. NMR Spectroscopy  NMR plot of a protein using 13C labelled material and NOE analysis
  34. 34. NMR Spectroscopy  Structure of the monomeric hemoglobin (MW = 16 kDa) using NMR spectroscopy – protein is shown in green and restraints in yellow
  35. 35. 3-Dimensional Electron Microscopy  Three dimensional electron microscopy (3D EM) works by focusing a beam of electrons and electron lenses on the protein and image it directly  The most commonly used technique involves imaging of many thousands of different single particles preserved in a thin layer of non-crystalline ice (cryo-EM)  Assuming each image captures the protein in a different orientation, a computational approach (similar to that used for CAT scans) will yield a 3D mass density map  With a sufficient number of single particles, the 3D EM maps can then be interpreted by fitting an atomic model of the macromolecule into the map  Recent advances in computer power has led to molecular and atomic detail approaching X-ray crystallography resolution (for 3D EM); cryo-EM has slightly lower resolution, showing protein domains and secondary structure
  36. 36. 3-Dimensional Electron Microscopy  As with NMR, a main advantage is avoiding the need to grow crystals  Sample preparation involves preservation in vitreous ice and then placing in the microscope (cryo-EM)  Used primarily on very large macromolecular structures where lower resolution is the norm  Combining with X-ray crystallography, NMR, mass spectrometry, fluorescence resonance energy transfer and computational techniques provides a way to view large structures in exquisite detail
  37. 37. 3-Dimensional Electron Microscopy  cryo-EM map of beta-galactosidase was built from over 90,000 images of the molecule frozen in ice
  38. 38. Cryo-Electron Microscopy Facility  The JEM-3200FS Field Emission Electron Microscope is equipped with a field emission electron gun of 300 kV accelerating voltage and an in-column energy filter  Equipment is made by a high- end speciality equipment company (JEOL)  Requires full time staff to run and maintain
  39. 39. Cryo-EM Structure of SARS-CoV-2 Spike (S) Protein (A) Schematic of SARS-CoV-2 S protein primary structure colored by domain. RBD domain (green color) encodes S protein domain. Arrows denote protease cleavage sites. (B) Side and top views of the prefusion structure of the SARS-CoV-2 protein with a single RBD in the up conformation. The two RBD down protomers are shown as cryo-EM density in either white or gray and the RBD up protomer is shown in ribbons colored corresponding to the schematic in (A).
  40. 40. Serial Femtosecond Crystallography  A free electron X-ray laser (XFEL) is used to create pulses of radiation that are extremely short (lasting only femtoseconds) and extremely bright  A stream of tiny crystals (nanometers to micrometers in size) is passed through the beam, and each X-ray pulse produces a diffraction pattern from a crystal, often burning it up in the process  A full data set is compiled from as many as tens of thousands of these individual diffraction patterns  Allows scientists to study molecular processes that occur over very short time scales, such as the absorption of light by biological chromophores
  41. 41. Growth of Structures in Protein Data Bank Year Number of PDB entries  Total number of X-ray, NMR, electron microscopy and modelled structures in PDB (yellow bars); blue bar is total number deposited per year
  42. 42. Protein Structure and Drug Discovery  The understanding of the structural and chemical binding properties of important drug targets in biologically relevant pathways can provide a unique advantage in discovering new drugs  Both empirical and computational methods are used to design and develop these drugs  Small molecule synthesis and testing  Antibody selection
  43. 43. Impacting Drug Discovery  Structural Biology is the application of protein structure technologies (e.g. X-ray crystallography, NMR, CryoEM) in identifying new drug therapies  This process is known as structure-based drug design (SBDD) Chemical Space Screening of Chemical Libraries Biological Space Finding New Targets Linked to Disease
  44. 44. Importance of Computational Methods in SBDD  Computational chemistry and biology are critically important in integrating theory and modelling with experimental observations  This is achieved by using algorithms, statistics and large databases  Simulates physical processes and uses statistics and data analysis to extract useful information from large bodies of data  Includes genomic and protein networks on the biology side and chemical/biochemical interactions and biophysical forces on the chemistry side  Of significant value to the biopharma industry as it helps (1) identify new disease targets (2) help understand the biology and what is needed to impact the disease and (3) creates new molecular entities (small molecule drugs, protein therapeutics, etc) that we can discover and develop to treat unmet medical need  Combining computational information and guidance with experimental data helps make the drug discovery process more efficient
  45. 45. Artificial Intelligence (AI) and Machine Learning (ML) in SBDD  Biology: Target identification within the protein network? What is the link to disease?  Experimental: Can I produce a structure? Can I produce a chemical library?  Chemistry: Can I optimize my compound to achieve the proper potency? Can I achieve the proper safety and selectivity?
  46. 46. Game Changer: From Primary Structure to 3D Structure  Deep Mind (UK-based AI company) has developed an algorithm that can predict the 3-dimensional shape of a protein (i.e. it’s tertiary structure) from its primary structure (i.e. amino acid sequence)  The algorithm, called AlphaFold, incorporates deep learning in which the software is trained on large data sets of sequences and structures to identify patterns that help determine the tertiary structure  Tested AlphaFold in the CASP (critical assessment of protein structure prediction) competition and was able to predict structures that matched experimental results Difficulty of protein structure prediction Global distance test % Easy Difficult AlphFold (2020)
  47. 47. Concepts Covered  Protein structure  Primary  Secondary  Tertiary  Quaternary  Protein folding  Protein structure determination  X-ray crystallography  NMR  Electron microscopy  Use of structure to design and develop new drug therapies