Research Interests | Current Projects | Future Projects
The unifying theme of my research is to combine structural and biochemical studies to investigate the macromolecular structure, function, and stability of biomolecules, especially of proteins implicated in human diseases. I am interested in studying how individual amino acids within the linear sequence of a native protein dictate its biochemical and biophysical character and how the specific structural elements within a given protein contribute to its function. My long-term scientific interest lies in exploiting this sequence-structure-function relationship in native proteins to develop strategies for the optimization of their biochemical and biophysical properties and to design enhanced proteins with specific functions.
Most of the proteins involved in important biological pathways are large multidomain proteins, which makes it a challenge to study them at a molecular level. Therefore, in my research, I use the protein dissection methodology, where such a multidomain protein is studied as an assembly of structurally independent, minimal functional units that can be characterized in isolation. My approach is biochemical for the in vitro solution characterization of the protein domain under investigation and biophysical in elucidating structure, conformational change, and binding. I utilize a wide spectrum of techniques such as chromatography, various spectroscopic methods including Fluorescence Spectroscopy, Nuclear Magnetic Resonance (NMR) Spectroscopy, Circular Dichroism, and X-ray crystallography, as well as other techniques such as Isothermal Calorimetry (ITC). I also use molecular biology to design and clone DNA constructs suitable for bacterial and mammalian expression systems.
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Despite the significant amount of research conducted in the field, one of the profound mysteries of biochemistry is still how the linear amino acid sequence encodes a fully folded and functional protein. In order to understand this “second half of genetic code”, we need many more detailed studies directed to understanding the forces that stabilize a native protein. The focus of the current projects in my lab is to provide a very detailed study and characterization of a small and independently folded polypeptide motif, called the Lin12/Notch Repeat (LNR).
LNRs were first described as three unique tandem modules crucial for the regulation of ligand induced proteolytic cleavage of the Notch receptor (1). More recently, they were identified within functionally unrelated multidomain proteins with different domain organizations, such as pregnancy associated proteins, and stealth proteins (2, 3). LNRs belong to a subset of small disulphide-rich protein folds (SDFs), which are among the most frequently utilized structural units in all of biology. However, LNRs are functionally different from most other SDFs since they participate in intramolecular regulation rather than intermolecular recognition. Based on the existing biochemical and structural information of a prototype LNR (4) and the protein database sequence alignment predictions, each LNR contains ~35-40 residues, has at least two disulphide bonds, and an absolute requirement for a Ca2+ ion to fold into its native structure with a very small amount of regular secondary structure.
Currently in my lab we are characterizing and comparing
LNRs from mainly the four different mammalian Notch proteins, as well as from other proteins
such as PAPP-A and Stealth Proteins. The two specific questions we are attempting to answer
through this work are:
- What is the inherent metal ion specificity and selectivity for the different LNRs?
- How does the number of predicted disulfides affect the folding and stability of the different the LNRs?
The overall goal for this research is to fill the existing gaps in our understanding of the sequence-structure-function relationship for these LNRs within the relevant specific biological background and also provide new insights into our global understanding of protein folding and stability.
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One of the long-term research goals for my lab is to utilize the findings and understanding obtained from current line of research in the lab to facilitate the redesign or de novo design of similar modules optimized for specific metal binding properties and protein stabilities. Therefore, as we accumulate sufficient data from current projects to guide us in the design of these optimized LNRs, there will be new projects in the lab that would involve varying degrees of protein engineering for different applications. One such application target of the lab will be to develop strategies for the use of these engineered LNRs as biochemical tools for the in vivo study of proteins containing these modules within their architecture, such as the Notch Receptors.