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Computer simulations of protein unfolding and ligand binding with enhanced sampling

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Ariane Ferreira Nunes Alves
Total Authors: 1
Document type: Doctoral Thesis
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
Defense date:
Examining board members:
Guilherme Menegon Arantes; Ernesto Raul Caffarena; Sandro Roberto Marana; Paulo Augusto Netz
Advisor: Guilherme Menegon Arantes

Molecular simulations may provide information and mechanistic insights that are difficult to obtain from experiments. However, biochemical phenomena such as ligand-protein binding and protein unfolding are slow and hard to sample on the timescales usually reached by conventional molecular dynamics (MD) simulations. These molecular phenomena were studied here by combining MD simulations with several methods or approximations to enhance configurational sampling: linear interaction energy (LIE) method, weighted ensemble (WE) approach and steered molecular dynamics (SMD). An equation was parametrized to predict affinities between small molecules and proteins based on the LIE approximation, which focus computational sampling in ligand bound and unbound states. Protein flexibility was introduced by using ensembles of configurations obtained from MD simulations. Different averaging schemes were tested to obtain overall affinities for ligand-protein complexes, revealing that many bound configurations contribute to affinities for flexible proteins, while affinities for rigid proteins are dominated by one bound configuration. T4 lysozyme (T4L) L99A mutant is probably the protein most often used to study ligand binding. Crystal structures show the artificial binding cavity created by the mutation has low accessibility, so protein movements or conformational breathing are necessary to allow the entry and egress of ligands. MD simulations were combined here with the WE approach to enhance sampling of infrequent benzene unbinding events from T4L. Four possible pathways were found and motions on alpha-helices and side chains involved in ligand egress were characterized. The four pathways correspond to protein tunnels previously observed in long MD simulations of apo T4L, suggesting that pathway heterogeneity along intrinsic tunnels is explored by small molecules to egress from binding cavities buried in proteins. Previous atomic force microscopy experiments revealed detailed information on the forced unfolding and mechanical stability of rubredoxin, a simple iron-sulfur protein. Complete unfolding of rubredoxin involves rupture of covalent bonds. Thus, the unfolding process was simulated here by SMD simulations coupled to a classical description of bond dissociation. Sampling of forced unfolding events was increased by using fast pulling velocities. Results were analyzed using a theoretical model valid for both slow and fast forced unfolding regimes. Simulations revealed that changing the points of force application along the rubredoxin sequence leads to different unfolding mechanisms, characterized by variable degrees of disruption of hydrogen bonds and secondary protein structure. (AU)

FAPESP's process: 14/17008-7 - Computer simulation of rare biochemical phenomena by enhanced sampling methods
Grantee:Ariane Ferreira Nunes Alves
Support Opportunities: Scholarships in Brazil - Doctorate