Yuba Bhandari

Exploring the diffusion of molecular oxygen in the red fluorescent protein mCherry using explicit oxygen molecular dynamics simulations.

The development of fluorescent proteins (FPs) has revolutionized cell biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for tagging and tracking cellular processes in vivo. Determining oxygen diffusion pathways in FPs can be important for improving photostability and for understanding maturation of the chromophore. We use molecular dynamics (MD) calculations to investigate the diffusion of molecular oxygen in one of the most useful monomeric RFPs, mCherry. We describe a pathway that allows oxygen molecules to enter from the solvent and travel through the protein barrel to the chromophore. We calculate the free-energy of an oxygen molecule at points along the path. The pathway contains several oxygen hosting pockets, which are identified by the amino acid residues that form the pocket. We also investigate an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.

Molecular dynamics investigations of the α-helix to β-barrel conformational transformation in the RfaH transcription factor.

The C-terminal domain (CTD) of the transcription antiterminator RfaH folds to an α-helix bundle when it interacts with its N-terminal domain (NTD) but it undergoes an all-α to all-β conformational transformation when it does not interact with the NTD. The RfaH-CTD in the all-α topology is involved in regulating transcription whereas in the all-β topology it is involved in stimulating translation by recruiting a ribosome to an mRNA. Because the conformational transformation in RfaH-CTD gives it a different function, it is labeled as a transformer protein, a class that may eventually include many other functional proteins. The structure and function of RfaH is of interest for its own sake, as well as for the value it may serve as a model system for investigating structural transformations in general. We used replica exchange molecular dynamics simulations with implicit solvent to investigate the α-helix to β-structure transformation of RfaH-CTD, followed by structural relaxation with detailed all atom simulations for the best replica. The importance of interfacial interactions between the two domains of RfaH is highlighted by the compromised structural integrity of the helical form of the CTD in the absence NTD. Calculations of free-energy landscape and transfer entropy elucidate the details of the RfaH-CTD transformation process.

Lattice model simulations of the effects of the position of a peptide trigger segment on helix folding and dimerization

The folding and dimerization of proteins is greatly facilitated by the presence of a trigger site, a segment of amino acids that has a higher propensity for forming α-helix structure as compared to the rest of the chain. In addition to the helical propensity of each chain, dimerization can also be facilitated by interhelical interactions such as saltbridges, and interfacial contacts of different strengths. In this work, we are interested in understanding the interplay of these interactions in a model peptide system. We investigate how these different interactions influence the kinetics of dimer formation and the stability of the fully formed dimer. We use lattice model computer simulations to investigate how the effectiveness of the trigger segment and its saltbridges depends on the location along the protein primary sequence. For different positions of the trigger segment, heat capacity and free energy of unfolded and folded configurations are calculated to study the thermodynamics of folding and dimerization. The kinetics of the process is investigated by calculating characteristic folding times. The thermodynamic and kinetic data from the simulations combine to show that the dimerization process of the model system is faster when the segment with high helical propensity is located near either end of the peptide, as compared to the middle of the chain. The dependence of the stability of the dimer on the trigger segments position is also studied. The stability can play a role in the ability of the dimer to perform a biological function that involves partial unzipping. The results on folding and dimer stability provide important insights for designing proteins that involve trigger sites.

Cooperative structural transitions in amyloid-like aggregation

Amyloid fibril aggregation is associated with several horrific diseases such as Alzheimers, Creutzfeld-Jacob, diabetes, Parkinsons, and others. Although proteins that undergo aggregation vary widely in their primary structure, they all produce a cross-β motif with the proteins in β-strand conformations perpendicular to the fibril axis. The process of amyloid aggregation involves forming myriad different metastable intermediate aggregates. To better understand the molecular basis of the protein structural transitions and aggregation, we report on molecular dynamics (MD) computational studies on the formation of amyloid protofibrillar structures in the small model protein ccβ, which undergoes many of the structural transitions of the larger, naturally occurring amyloid forming proteins. Two different structural transition processes involving hydrogen bonds are observed for aggregation into fibrils: the breaking of intrachain hydrogen bonds to allow β-hairpin proteins to straighten, and the subsequent formation of interchain H-bonds during aggregation into amyloid fibrils. For our MD simulations, we found that the temperature dependence of these two different structural transition processes results in the existence of a temperature window that the ccβ protein experiences during the process of forming protofibrillar structures. This temperature dependence allows us to investigate the dynamics on a molecular level. We report on the thermodynamics and cooperativity of the transformations. The structural transitions that occurred in a specific temperature window for ccβ in our investigations may also occur in other amyloid forming proteins but with biochemical parameters controlling the dynamics rather than temperature.