Proteins are dynamic molecules. Under native conditions, protein molecules are in thermodynamic equilibrium with transiently unfolded conformations. Understanding the nature of this transient unfolding event is one of the central questions in protein biophysics. Structures of partially-unfolded conformations transiently populated under native conditions provide important clues in elucidating protein folding mechanisms. Information on transient partial unfolding is also critical in understanding the mechanisms of protein aggregation, misfolding, and degradation. The intellectual merit of this project is to investigate the rare but critical events of transient partial unfolding proteins undergo in native conditions to further the understanding of how conformation energy landscapes control function and regulation. The specific research objectives are (1) to test the cooperativity of alpha-helix unfolding in the context of a folded protein under native conditions, (2) to test if transient partial unfolding in bacterial periplasmic proteins is a consequence of the conformational energy landscapes optimized for vectorial folding during translocation, and (3) to engineer robust proteins by utilizing information on transient partial unfolding.
The broader impact of this project is education and training of graduate and undergraduate students on the principles of molecular biophysics and their application to real-world problems. First, through participation in this project, graduate and undergraduate students will have a valuable opportunity to learn the basic principles of protein biophysics and biochemistry. Students will be trained in protein engineering, protein expression and purification, and biophysical characterization of proteins through hands-on experience. Second, the results and ideas generated by this project will be directly applied to the development of a new course on biotechnological applications of proteins and peptides. The conformational equilibrium of protein structures, including protein folding, is an excellent topic to expose students to the basic principles of thermodynamics and kinetics and their applications to practical problems. Moreover, findings from this project will provide students with vivid examples of the relevance of protein conformations to practical challenges in the development and application of protein products.
Many proteins require their native structure to perform their biological functions. Loss of the structure results in inactivation of the proteins. Even under native conditions in which proteins normally function, proteins transiently lose their structures mostly through partial unfolding. In this project, we investigated how frequently proteins experience transient partial unfolding and what structural changes occur during partial unfolding using proteolysis as a probe. Proteases hydrolyze peptide bonds in proteins only when the bond is exposed to solvent. Exploiting this property, we elucidate the thermodynamics of transient partial unfolding. First, we investigated partial unfolding of E. coli dihydrofolate reductase (DHFR). 1) We determined the structure of the partially unfolded form of and demonstrated that the partially unfolded form is structurally homologous to an obligatory folding intermediate of the protein. The detailed structure of the partially unfolded form provided an insight on the final step of the folding of DHFR. 2) We investigated the effect of NADP+ on partial unfolding of DHFR and found that DHFR can unfold partially even in the presence of NADP+. Still, partial unfolding of DHFR is suppressed in the presence of NADP+ because NADP+ has a reduced affinity to the partially unfolded form. 3) We investigated the effect of circular permutation on partial unfolding of DHFR. Circular permutation modifies the chain connectivity of a protein without altering the three-dimensional structure. We found that partial unfolding of DHFR is promoted when new termini are introduced in proximity of the unfolded region of the partially unfolded form. Second, we investigated the role of a salt bridge in partial unfolding of E. coli ribonuclease H (RNase H). A salt bridge is a pair of positively- and negatively-charged residues in protein structure. We found that this salt bridge in RNase H does not affect the global stability of the protein but suppresses partial unfolding of the protein. This result suggests a previously unknown role of salt bridges in protein structure. Third, we investigated a role of a metabolite in stabilizing a partially unfolded form of E. coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We demonstrated that ATP does not interact with the tetrameric native form of GAPDH but stabilizes selectively its dimeric folding intermediate. We also show ATP facilitates significantly the folding of the protein. This study demonstrates an unprecedented role of a metabolite in protein folding. Fourth, we investigated the transition-state structure of an α-helical membrane protein, bacteriorhodopsin (bR). Despite their many critical roles in biology, folding of membrane proteins is significantly less investigated than soluble proteins. Hydrolysis of the retinal cofactor from bR in the unfolded state complicates the kinetic study of folding and unfolding of this protein. We developed a kinetic model to extract information on the folding and unfolding kinetics from the complex kinetics. Using the kinetic model, we discovered that the structure of bR is poorly organized in its transition state. We have reported these results in three research articles already. Two manuscripts are currently under review, and two more manuscripts are being prepared. We have presented our findings in several international research conferences. We also trained five graduate students and seven undergraduate students on this project. Two of the undergraduate students became authors of our manuscripts.
