I am interested in understanding how changes in a protein’s conformation affect its function, and the current focus of my research group is on understanding the mechanisms by which proteins fold. This is part of a long-standing basic research question in Biophysics: If we know a protein’s amino acid sequence, can we predict its structure and its function? Further, many diseases, such as Alzheimer’s, Huntington, and Jakob Creutzfeldt in humans and mad cow and wasting disease in animals are at least partially caused by misfolded proteins, so an understanding of how proteins fold will have implications in finding cures. The ultimate goal would be to understand the actual sequence of motions and events that occur as a protein moves from a denatured, extended state in solution to the compact and correctly folded conformation needed for it to properly function. Towards reaching this goal, we are seeking to understand how protein folding mechanisms are changed by constraints in available volume (the cell cytoplasm is very crowded) and the presence of ions and crowding agents (large bulky molecules).
In the cell, proteins are synthesized on the ribosome, and as the protein polymer forms, it begins to fold. Complete folding may not proceed very far until the full protein is synthesized, but in general folding is very efficient—proteins find their native state quickly. The first step in this process is a rapid collapse from an extended structure into a compact, but not fully folded intermediate, sometimes called the “molten globule.” Some of the secondary structure elements, such as helices and sheets, may be partially formed during this collapse, but the final fold is only achieved from within this molten globule.
Sol-gel encapsulation techniques: A significant part of our research effort is to understand what motions are available to proteins once they attain a compact conformation. It is not possible to force a protein to remain compact in bulk solution because there is nothing to constrain it to remain within a small volume. However, a protein that is beginning to unfold can be so constrained if we encapsulate it within a sol-gel glass. When mixed with water, tetramethyl orthosilicate (TMOS) polymerizes to form a porous SiO2 glass around proteins in solution such that the protein retains its biological function. Diffusive connectivity via the gel pores is maintained between the encapsulated protein and the solvent so different pH buffers, substrates, and redox agents can be added to the proteins via the liquid phase in which the gel is immersed. These added agents can be used to trigger unfolding or refolding. We were the first group to demonstrate that a protein, myoglobin, could be unfolded and then refolded within the sol-gel pores (Peterson, et al., 2008. Biophysical Journal, 95: 322-332), and our current work is examining how different ions influence folding within the gel pores.
In the 1880’s, Hofmeister ranked several ions by their ability to facilitate folding, subunit assembly, crystallization, and aggregation of proteins:
Anions: HPO4-2 ~ SO4-2 > F- > acetate > Cl- > Br- > NO3- > I- > ClO4- > SCN-
Cations: K+ = Na+ > Li+ > NH4+ >> GdnH+
In addition, the effects of these ions are additive. We are currently exploring how these ions stabilize/destabilize the folded state vs. the unfolded and partially folded states in the folding pathway of small heme proteins.
In a typical experiment, we start with the proteins encapsulated in their folded state, then add the ions that we wish to test in terms of their influence on protein structure. Next, we add a denaturant to initiate unfolding (guanidinium, urea, low or high pH buffers, or increases in temperature) and monitor the protein structure as a function of time. We will use UV/Vis absorption spectroscopy in the initial experiments. I am currently assembling a time-resolved Raman apparatus that will incorporate nanosecond lasers, and we will use this to follow protein motions within both the gels and solution in real time.