- Protein-protein interactions of HIV-I [All]
- Shiftides: peptides that shift the oligomerization state of proteins [Representative Paper] [All]
- Biophysical studies of apoptosis related proteins
- Natively unfolded proteins. [ASPP2] [C-Vif]
The concept:Our group is interested in the quantitative biophysical analysis of protein-protein interactions in health and disease, and the use of the data as a basis for rational drug design. We are looking at biological systems that are affected in disease, such as cancer-related pathways in the cell, or viral infection. The research involves three major steps:
- Analyzing protein-protein interactions in health, to understand how the particular biological system works at the molecular level.
- Understanding what goes wrong at the molecular level in disease, e.g. upon mutation.
- Development of drugs that will restore the biological system to its healthy conditions (in cancer), or will inhibit undesired interactions (in viral infection).
- Peptide chemistry
- Protein biochemistry (expression and purification)
- Biophysical studies (such as fluorescence and NMR)
- Biophysical studies of protein-protein interactions at the molecular level, and of the effects of mutations and post-translational modifications on these interactions.
- Biophysical studies of the p53-binding protein ASPP2, its protein-protein interactions and its regulation.
- "Chemical chaperones" - Development of peptides and small molecules that assist in refolding of proteins whose misfolding lead to disease.
- Studies of protein-protein interactions of HIV-1 proteins, and development of lead anti-viral compounds that inhibit these interactions.
What techniques are we using?
- The research on each biological system will involve 3 major parts:
- The biochemical part: expression and purification of the proteins of interest.
- The chemical part: synthesis of peptides, peptidomimetics and - later on - small organic molecules.
- The biophysical part: studies of the protein-protein, protein-peptide and protein-DNA interactions using fluorimetry, UV spectroscopy, NMR, CD and other biophysical methods.
Why study biological systems at the molecular level?
Biology is the chemistry of the non-covalent bond.
The living cell is a dynamic complex system, where biological processes are mediated by changes in non-covalent interactions between bio-molecules (such as proteins and nucleic acids). Understanding these interactions at the molecular level is crucial for our understanding of how the living cells function. Moreover, impairment of protein-protein and protein-DNA interactions (e.g. due to mutations) can lead to severe diseases, such as cancer. Understanding the effects of mutation on protein-protein interactions at the molecular level could provide the background for the development of drugs that will restore the impaired interactions. In cases of viral infection, understanding the mechanisms at the molecular level is crucial for the development of anti-viral lead compounds.
We are doing both: studying the biological systems, and developing lead compounds.
Why using biophysics?
One of the main goals of modern biology is to understand the relationship between molecular structure and biological function. In the post-genomic era, a huge amount of structural and molecular information about protein-protein interactions is available in the databases. Cell biology provides general information about these interactions in the cellular environment, and X-ray crystallography has provided a wealth of information about the structures of macromolecules. However, these approaches do not provide quantitative information about the biological interaction of interest, and cannot describe the dynamics of biological systems. As biological processes are mediated by changes in non-covalent molecular interactions, the biological functions of macromolecules are defined by the kinetics and energetics of these changes. Hence, a combined analysis of structure and dynamics allows a detailed and quantitative description of the biological system.
Biophysical methods are uniquely suited to study the thermodynamics and kinetics of interactions between macromolecules at the molecular level. Various biophysical techniques are available to characterize binding and assembly of macromolecules in solution, including calorimetry, fluorescence spectroscopy, UV spectroscopy, stopped-flow kinetics, circular dichroism and NMR. These methods can provide the accurate quantitative information about the binding affinity between macromolecules such as proteins and nucleic acids. They also allow us to explore the interaction on the molecular level and obtain parameters such as the order of the binding kinetics, the kinetic rate constants and the thermodynamics of the interactions (including independent measurements of G, S and H). NMR gives information about the dynamic structural changes that take place in the macromolecules upon interaction. Chemical and thermal denaturation experiments can teach us about protein stability and about how various ligands affect it.
The information we can obtain using biophysical studies (a partial list):
- Measure binding affinities (dissociation constants) between proteins and between proteins and their ligands.
- Measure which residues are important for binding and how mutation affects binding
- Identify protein-binding and ligand-binding sites in proteins using methods such as HSQC NMR and peptide mapping.
- Measure the thermodyanmic stability of proteins and how modifications and mutations affect it.
- A combination of peptide synthesis and biophysical measurements enables us to study the effect of specific post-translational modifications (such as phopshorylations) in proteins on the binding to partner proteins.
- Measure the kinetics of protein-protein interactions.
The use of peptides to study protein-protein interactions:Our lab is using peptides as a major tool to study protein-protein interactions. This has several advantages:
- To study isolated domains from proteins.
- To study proteins which are difficult to express and purify in sufficient amounts.
- Synthesis and purification is easy and in large amounts, and is done in-house.
- To focus on particular sites in proteins, and identify binding regions
- To study post-translational modifications (phosphorylation, acetylation). Peptide synthesis is the only way to introduce specific modifications in the desired positions in a protein sequence
- When we need to introduce specific fluorescent (or other) labels in the protein sequence.
Chemical synthesis is a huge advantage, especially when we want to introduce modifications at specific locations in the sequence with 100% yield and specificity, and in order to avoid the problems of expression in bacterial systems, and get large amounts of pure material in high yield