We have made significant progress in several areas related to protein dynamics, folding, binding, and function.? ? Multi-protein assemblies: We developed coarse-grained models and effective energy functions for? simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (Kd > 1 micromolar). Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair? potentials and DebyeHckel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. The energy functions was parametrized against protein osmotic second virial coefficients, and validated against protein binding affinities. We could show that both binding affinities and complex structures are in good agreement with experiment. With the validated model, we simulated the interaction between the Vps27 multiprotein complex of the ESCRT membrane-protein trafficking system and a membrane-tethered ubiquitin. We found that the specific and nonspecific interactions of the complex and ubiquitin are positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the ∼300 micromolar M of the specific domains. We also found that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.? ? Transient encounter complexes in protein association. Recent paramagnetic relaxation enhancement (PRE) studies on several weakly interacting protein complexes have unequivocally demonstrated the existence of transient encounter complexes. In collaboration with Drs. Tang and Clore (NIDDK, NIH), we used the coarse-grained simulation model developed for studies of multiprotein assemblies to study transient protein binding. Specifically, we conducted a joint analysis of simulation and experiment to explore the physical nature and underlying physicochemical characteristics of encounter complexes involving three proteinprotein interactions of the bacterial? phosphotransferase system. Replica-exchange Monte Carlo simulations using a coarse-grained energy function recovered the structures of the specific complexes and produced binding affinities in agreement with experiment. Together with the specific complex, a relatively small number of distinct nonspecific complexes largely accounts for the measured PRE data. The combined? relative population of the latter is less than 10%. The binding interfaces of the specific and nonspecific complexes differ primarily in size but exhibit similar amino acid compositions. We found that the overall funnel-shaped energy landscape of complex formation is dominated by the specific complex, a small number of structured nonspecific complexes, and a diffuse cloud of loosely bound complexes connecting the specific and nonspecific binding sites with each other and the unbound state. Nonspecific complexes may not only accelerate the binding kinetics by enhancing the rate of success of random diffusional encounters but also play a role in protein function as alternative binding modes.? ? Cholesterol transport: In a series of equilibrium and nonequilibrium molecular dynamics simulations, done in collaboration with Drs. Canagarajah, Hurley, and Prinz in NIDDK, NIH, we explored the dynamics in the cholesterol transporter Osh4, a member of the family of oxysterol-binding proteins. We could show the cholesterol release was associated with the opening of a lid covering the cholesterol binding site. The exit from the binding pocket proceeded in steps involving (i) the breaking of water-mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.? ? Signal transduction: Transcription factors are central components of the intracellular regulatory networks that control gene expression. In a series of computer simulations, done in collaboration with Drs. Gutkind and Turjanski at NIDCR, NIH, and Dr. Best at University of Cambridge, we studied the folding and binding of the natively-unstructured phosphorylated pKID domain of the transcription factor CREB to the KIX domain of the co-activator CBP. Our simulations of a topology-based model predicted a coupled folding and binding mechanism, and the existence of partially bound intermediates. From transition-path and phi-value analyses, we concluded that the binding transition state resembles the unstructured state in solution, implying that pKID of CREB becomes structured only after committing to binding. ncreasing the amount of structure in the unbound pKID reduces the rate of binding. Our study helps explain how being unstructured can confer an advantage in protein target recognition, and provides new insights how signal transduction is accomplished at the molecular level.? ? G-protein coupled receptor dynamics. In collaboration with Drs. Tikhonova, Gershengorn, and Costanzi in NIDDK, NIH, and Dr. Best at the University of Cambridge, we studied the dynamics of rhodopsin, which serves as a prototypical G protein-coupled receptor (GPCR). With the help of molecular dynamics and coarse-grained modeling, we used experimental distance information and x-ray structures to develop a structural model of the META II intermediate. We then simulated rhodopsin activation in a dynamic model to study the path leading from LUMI to our META II model for wild-type rhodopsin and a series of mutants. The simulations showed a strong correlation between the transition dynamics and the pharmacological phenotypes of the mutants. These results help identify the molecular mechanisms of activation in both wild type and mutant rhodopsin. Such dynamic models of activation should be applicable to study the pharmacology of other GPCRs and their ligands, offering a key to predictions of basal activity and ligand efficacy.? ? Peptide structure and dynamics. In a series of papers, done in collaboration with Dr. Best (formerly at NIDDK, NIH and now at University of Cambridge, UK) and Dr. Buchete (formerly at NIDDK, NIH and now at University College Dublin, Ireland), we studied the structure, dynamics and folding of short alanine-based peptides. From these studies we gained important new insights into the quality of current molecular force fields, by comparing the simulations to state-of-the-art NMR experiments. This comparison points the way toward improved simulation models, a key step to making biomolecular simulations a quantitatively accurate tool in biophysical studies. We also made major methodological advances. We could show that a master-equation framework allows us to extract both thermodynamic and kinetic properties of pepide folding from molecular simulations. The master equation models not only give access to the slow conformational dynamics but also shed light on the molecular mechanisms of folding.
| Rosta, Edina; Buchete, Nicolae-Viorel; Hummer, Gerhard (2009) Thermostat artifacts in replica exchange molecular dynamics simulations. J Chem Theory Comput 5:1393-1399 |
| Best, Robert B; Hummer, Gerhard (2009) Biochemistry. Unfolding the secrets of calmodulin. Science 323:593-4 |
| Kim, Young C; Tang, Chun; Clore, G Marius et al. (2008) Replica exchange simulations of transient encounter complexes in protein-protein association. Proc Natl Acad Sci U S A 105:12855-60 |
| Best, Robert B; Buchete, Nicolae-Viorel; Hummer, Gerhard (2008) Are current molecular dynamics force fields too helical? Biophys J 95:L07-9 |
| Buchete, Nicolae-Viorel; Hummer, Gerhard (2008) Peptide folding kinetics from replica exchange molecular dynamics. Phys Rev E Stat Nonlin Soft Matter Phys 77:030902 |
| Buchete, Nicolae-Viorel; Hummer, Gerhard (2008) Coarse master equations for peptide folding dynamics. J Phys Chem B 112:6057-69 |
| Kim, Young C; Hummer, Gerhard (2008) Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding. J Mol Biol 375:1416-33 |
| Canagarajah, Bertram J; Hummer, Gerhard; Prinz, William A et al. (2008) Dynamics of cholesterol exchange in the oxysterol binding protein family. J Mol Biol 378:737-48 |
| Tikhonova, Irina G; Best, Robert B; Engel, Stanislav et al. (2008) Atomistic insights into rhodopsin activation from a dynamic model. J Am Chem Soc 130:10141-9 |
| Turjanski, Adrian Gustavo; Gutkind, J Silvio; Best, Robert B et al. (2008) Binding-induced folding of a natively unstructured transcription factor. PLoS Comput Biol 4:e1000060 |
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