Department of Biological Sciences
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Item G-Quadruplex Structure Can Be Stable with Only Some Coordination Sites Being Occupied by Cations: A Six-Nanosecond Molecular Dynamics Study(ACS, 2001) Chowdhury, ShibasishGuanine tetrads are formed spontaneously by guanine rich sequences in the presence of certain cations. Various quadruplex helical structures, stabilized by such tetrads, apparently play an important biological role in vivo. To understand the importance of the cations, a 6 ns molecular dynamics simulation has been performed on a 7-mer G-quadruplex, surrounded by Na+ counterions and explicit water molecules, but without any ions in the initial structure. Interestingly, the quadruplex structure does not fall apart, but undergoes small structural changes, which enable the solvent molecules, including Na+ ions, to enter the empty central channel of structure. This channel is fully hydrated within the first 100 ps and two ions move into the central channel between 0.5 and 2 ns of MD simulation, by replacing some of the water molecules. The ions once trapped within the quadruplex channel are not expelled even during 1.5 ns of MD at 400 K. In fact they penetrate deeper into the channel to facilitate entry of additional ions, though all coordination sites within the quadruplex are not occupied even after 6.1 ns of MD simulation. The entry of cations into the central channel leads to a quadruplex structure with more favorable free energy of hydration, which is comparable to that of a fully coordinated quadruplex.Item Characterizing the rate-limiting step of Trp-cage folding by all-atom molecular dynamics simulations(ACS, 2004) Chowdhury, ShibasishIn this study, the detailed mechanisms of the rapid-folding Trp-cage mini-protein were investigated by extensive all-atom molecular dynamics simulations of both wild-type and mutant proteins using a recently developed point-charge force field within the AMBER simulation package and the generalized Born treatment of solvation. Among the 77 100-ns simulations performed on the wild-type protein, 5 of the simulation trajectories yielded structures with main-chain RMSDs of 1.0−2.0 Å from the native NMR structure. A gradual reduction in the value of the main-chain RMSD distribution was observed during the simulations, which is consistent with the folding funnel theory. The folding time of ∼3 μs based on native tertiary contacts is in reasonable agreement with an experimental value of ∼4 μs. Detailed analysis suggests that packing of the structurally important Trp25 side chain is involved in the rate-limiting step and unfolding of the misfolded states and overcoming the additional entropic barrier also contributed to the rate-limiting steps. This is reinforced by the faster folding rate of the W25F mutant. Two putative folding pathways were observed from the simulations, and their folding rates differed by about 200-fold, leading to a 3.2 kcal/mol folding free energy barrier difference. Of this, approximately 2.2 kcal/mol was due to unfolding of the misfolded states, and about 1.0 kcal/mol was due to overcoming the entropic cost to move Trp25 side chain into the native orientation. Although formation of the main-chain contacts was not the rate-limiting step, we observed a hierarchical process in which the short-range native contacts formed faster than the long-range ones. These observations are consistent with the contact-order theory.Item Denatured-state ensemble and the early-stage folding of the G29A mutant of the B-domain of protein A(ACS, 2005-04) Chowdhury, ShibasishThe folding mechanism of the G29A mutant of the B-domain of protein A (BdpA) has been studied by all-atom molecular dynamics simulation using AMBER force field (ff03) and generalized Born continuum solvent model. Started from the extended chain conformation, a total of 16 simulations (400 ns each) at 300 K captured some early folding events of the G29A mutant of BdpA. In one of the 16 trajectories, the G29A mutant folded within 2.8 Å (root mean square) of the wild-type NMR structure. We observed that the fast burial of hydrophobic residues was the driving force to bring the distant residues into close proximity. The initiation of the helix I and III occurred during the stage of hydrophobic collapse. The initiation and growth of the helix II was slow. Both the secondary structure formation and the development of the native tertiary contacts suggested a multistage folding process. Clustering analysis indicated that two helix species (helices I and III) could be intermediates. Further analysis revealed that the hydrophobic residues of partially folded helix II formed nativelike hydrophobic contacts with helices I and III that stabilized a nativelike state and delayed the completion of folding of the entire protein. The details of the early folding process were compared with other theoretical and experimental studies. It was found that a nativelike hydrophobic cluster was formed by residues including F30, I31, L34, L44, L45, and A48 that prevented further development of the native structures, and breaking the hydrophobic cluster like this one contributed to the rate-limiting step. This was in complete agreement with the recent kinetic measurements in which mutations of these residues to Gly and Ala substantially increased the folding rates by as much as 60 times. Apparently, destabilization of nonnative states dramatically enhanced the folding rates.