Molecular Dynamics Simulations of Cross-Linked Peptides and Enzymes

Abstract

Protein engineering typically utilizes amino acid mutations within a protein sequence to enhance activity, stability, or selectivity. We present a new approach to protein engineering by incorporating cross-linkers at pre-determined locations in the protein structure, a method we term Site-Directed Cross-Linking (SDXL). SDXL is a promising strategy for studying the structure, function, and dynamics of enzymes. Together, cross-linking and mutation studies offer great insight into the fundamental biophysics of enzymatic systems.We first applied this method to a set of model helical peptides in order to build a predictive computational model of reaction rates for ring-closure cross-linking reactions. Jacobsen-Stockmeyer theory predicts that entropy is the sole rate-determining factor for ring-closure reaction rates. By computing the difference in conformational entropy between the fully cross-linked peptide and the uncross-linked mono-adduct, we are able to reproduce the trends in experimentally measured cross-linking reaction rates in the set of model helical peptides. These trends highlight the helical structure of the model peptides and demonstrate the potential of SDXL for probing the secondary and tertiary structure of larger, more complex, proteins.Dihydrofolate reductase (DHFR) is an enzyme that is critical for the biosynthesis of purines. As such it is a common target for antibiotics, cancer therapies, and antimalarial drugs. We have initiated SDXL studies on the e. coli DHFR to investigate whether it is possible to modulate enzyme activity in a rational and controlled way. A single cross-linker was designed and introduced by SDXL between the active-site M20 loop and the distal FG loop that is essential for stabilizing the closed (and active) M20 conformation. We observe how the perturbations introduced by various forms of the cross-linker change the dynamics of the protein, and correlate these structural and dynamic changes with enzyme activity.