MOLECULAR CELL BIOLOGY

1. PROTEINS, DNA AND
CHROMOSOMES
MODULE 1
-Haymanti Bhanot, PhD

2. PROTEIN
FUNCTION
MODULE 1: LESSON 2 OF 3
BIOL6299 Fall 2022
Northeastern University
(Source: Bruce Alberts; Alexander Johnson; Julian Lewis; David Morgan; Martin Raff; Keith Roberts; Peter Walter: 6th Edition)

3. INTRODUCTION
• In this lesson, we discuss how proteins bind to specific molecules and how their
activity determined protein function in the cell.
• By the end of this lesson, you will have the opportunity to:
• Explain the various types of protein binding.
• Discuss how enzymes are regulated within the cells
• Describe major protein mechanisms and modifications that have major impacts in cell
signaling and regulation.
• Identify common methods used to analyze proteins and their cellular roles.

4. MODULE 1
LESSON 2
OF 3
•Protein Specificity
•Binding site of Proteins
•Types of protein binding
•Role of enzymes
Protein binding and the role of Enzymes
•Feedback inhibition
•Allosteric Regulation
•Protein modifications and their role in protein regulation
Protein Regulation
•Yeast two hybrid, Bioinformatics, Affinity Tags, Proteomics,
Protein Mass Spectrometry
Methods to Analyze Proteins

5. PROTEIN BINDING AND
THE ROLE OF ENZYMES
BIOL6299 Spring 2022
Northeastern University

6. PROTEIN SPECIFICITY
Ø Ligand – molecule bound by
a protein, the binding site of
which is often located in the
cavity of the protein.
Ø Selectivity and affinity is
dependent upon the
noncovalent bonds.

7. THE BINDING SITE OF A PROTEIN
Ø The folding of the polypeptide chain creates
a cavity on the protein surface.
Ø This cavity consists of a set of amino acid
side chains arranged to form noncovalent
bonds only with certain ligands.

8. TYPES OF PROTEIN BINDING

9. PROTEIN-LIGAND
INTERACTION -
ANTIBODIES
• Antibodies are proteins generated in response
to foreign invaders.
• To be effective, each Ab must bind tightly to a
specific antigen, tagging it for destruction by
other immune cells.

10. THE ROLE OF
ENZYMES
• Enzymes are proteins that bind substrates and
convert them into other chemicals called
products.
• Enzymes have higher affinity for unstable
transition states of substrate than for stable
form.
• Enzymes reduce the activation energy and act
as catalysts to speed up reactions.

11. STRATEGIES OF ENZYME CATALYSIS
HOLDING SUBSTRATES CHARGE STABILIZATION APPLYING FORCES

12. CATALYSIS BY LYSOZYMES
Ø LYSOZYME REACTION INVOLVES HYDROLYSIS.
Ø LYSOZYMES CATALYZES THE CLEAVAGE OF SPECIFIC COVALENT BOND IN THE POLYSACCHARIDE BACKBONE CHAIN AND
SEVERS THE CHAIN.

13. PROTEIN REGULATION
BIOL6299 Spring 2022
Northeastern University
• ALLOSTERI C PROTEI NS
• KI NASES
• PHOSPHORYLATI ON
• SCAFFOLDI NG

14. PROTEIN REGULATION – FEEDBACK
INHIBITION
• Enzyme activity is regulated by 4 distinct mechanisms-
• Controlling the level of gene expression of each enzyme.
• Confining sets of enzymes to particular subcellular compartments.
• Covalent modifications of the protein itself.
• Regulation by a molecule other than the substrate.
• Feedback inhibition-
• Involves substrate binding to the enzyme at a special regulatory site outside the active site,
thereby altering the rate by which the enzyme converts substrate to products.

15. FEEDBACK
INHIBITION
Positive feedback – stimulates
enzyme activity
Negative feedback – inhibits
enzyme activity (mostly of an
enzyme that acts earlier in the
pathway)

16. ALLOSTERIC
REGULATION –
ASPARTATE
TRANSCARBAMOYLASE
Pyrimidine biosynthesis
High concentrations of CTP
deactivate the enzyme in a
negative feedback mechanism.

17. REGULATION
OF ENZYME
BY
MOLECULES •Reversible
•Irreversible
Competitive inhibitors
•Inhibitors
•Activators
Allosteric

18. ALLOSTERIC ENZYMES
Allostery is a change in a
protein’s conformation brought
about by the binding of a
regulatory ligand

19. PROTEIN PHOSPHORYLATION
Addition of a
phosphate can either
activate or inhibit
enzyme activity.

20. KINASES – CYCLIN
DEPENDENT
KINASES
• CDKs are serine and
threonine kinases
• Required for normal cell-
cycle control.
• There are 3 signals/inputs
required for fully functional
Cdk.

21. KINASES – SRC KINASES
Src protein is a tyrosine kinase.
Through tyrosine phosphorylation, they transmit intracellular signals from the receptor itself.
It has 3 main domains on the Src protein:
2 peptide binding molecules- SH3 and SH2
1 catalytic kinase domain- SH1

22. KINASES – SRC KINASES CONTD…
Two signals are required to activate Src kinase:

23. INDIRECT PHOSPHORYLATION -RAS
PROTEINS (GTP)
Ras protein GAPs and GEFs

24. GTPASE – MOLECULAR SWITCHES
* Phosphorylation can also regulate proteins as part of the guanine nucleotide GTP,
which also binds tightly to the protein.

25. THE SCAFFOLDING
PROTEINS: SCF
UBIQUITIN LIGASE
*Proteins can also form complexes that serve as large
protein machines.
E.g.: SCF ubiquitin ligase (has 5 subunits) binds
different target proteins and covalently attaches a
ubiquitin polypeptide, which tags the protein for
destruction.

26. POST TRANSLATIONAL MODIFICATIONS
Ø A large number of PTMs also regulate protein structure and function.

27. PROTEIN MODIFICATIONS: P53
Ø Combinatorial Regulatory role: Proteins can be modified at multiple amino acids.
Ø E.g.: p53 (tumor suppressor gene – apoptosis, cell cycle and damage response).
Ø P53 can be regulated by many different combinations of PTMs at 20 different sites.

28. METHODS TO ANALYZE
PROTEINS
BIOL6299 Spring 2022
Northeastern University
• YEAST TWO HYBRI D
• BI OI NFORMATI CS
• AFFI NI TY TAGS
• PROTEOMI CS
• PROTEI N MASS SPECTROMETRY

29. YEAST TWO
HYBRID SYSTEM
• Focuses on uncovering protein-protein
interactions that occur in the cell

30. BIOINFORMATICS
• Uses many areas of computer science,
mathematics and engineering to process
biological data, to develop software tools to
generate useful biological knowledge.

31. AFFINITY TAGS
• Affinity tags are appended to proteins so that
they can be purified from their crude biological
source using an affinity technique.
• Tags: His, GFP, Myc, etc.
• These tags are useful for immunoblotting,
immunofluorescence and immunoprecipitation
experiments.

32. PROTEOMICS
• Proteomics is a large scale study of proteins, particularly
their structure and function.
• Proteome is the entire compliment of proteins, including
modifications made to a particular set of proteins,
produced by an organism or system (varies with time and
distinct requirements or stresses, that a cell or organism
undergoes).

33. PROTEIN MASS
SPECTROMETRY
• Important for characterization of proteins.
• 2 primary methods used for ionization of whole proteins
are:
• Electrospray ionization (ESI)
• Matrix assisted laser desorption/ionization (MALDI)

34. IN THIS LESSON:
• Learned about the various types of protein binding
• Discussed how enzymes are regulated within the cells.
• Described major protein mechanisms and modifications that have major impacts
in cell signaling and regulation.
• Identified common methods used to analyze proteins and their cellular roles.