BackgroundPopular bioinformatics approaches for studying protein functional dynamics include comparisons of crystallographic structures, molecular dynamics simulations and normal mode analysis. However, determining how observed displacements and predicted motions from these traditionally separate analyses relate to each other, as well as to the evolution of sequence, structure and function within large protein families, remains a considerable challenge. This is in part due to the general lack of tools that integrate information of molecular structure, dynamics and evolution.ResultsHere, we describe the integration of new methodologies for evolutionary sequence, structure and simulation analysis into the Bio3D package. This major update includes unique high-throughput normal mode analysis for examining and contrasting the dynamics of related proteins with non-identical sequences and structures, as well as new methods for quantifying dynamical couplings and their residue-wise dissection from correlation network analysis. These new methodologies are integrated with major biomolecular databases as well as established methods for evolutionary sequence and comparative structural analysis. New functionality for directly comparing results derived from normal modes, molecular dynamics and principal component analysis of heterogeneous experimental structure distributions is also included. We demonstrate these integrated capabilities with example applications to dihydrofolate reductase and heterotrimeric G-protein families along with a discussion of the mechanistic insight provided in each case.ConclusionsThe integration of structural dynamics and evolutionary analysis in Bio3D enables researchers to go beyond a prediction of single protein dynamics to investigate dynamical features across large protein families. The Bio3D package is distributed with full source code and extensive documentation as a platform independent R package under a GPL2 license from http://thegrantlab.org/bio3d/.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0399-6) contains supplementary material, which is available to authorized users.

For simulating proteins at work in millisecond time scale or longer, we develop a coarse-grained (CG) molecular dynamics (MD) method and software, CafeMol. At the resolution of one-particle-per-residue, CafeMol equips four structure-based protein models: (1) the off-lattice Go model, (2) the atomic interaction based CG model for native state and folding dynamics, (3) the multiple-basin model for conformational change dynamics, and (4) the elastic network model for quasiharmonic fluctuations around the native structure. Ligands can be treated either explicitly or implicitly. For mimicking functional motions of proteins driven by some external force, CafeMol has various and flexible means to "switch" the energy functions that induce active motions of the proteins. CafeMol can do parallel computation with modest sized PC clusters. We describe CafeMol methods and illustrate it with several examples, such as rotary motions of F1-ATPase and drug exports from a transporter. The CafeMol source code is available at www.cafemol.org .

Bio3D is a family of R packages for the analysis of biomolecular sequence, structure, and dynamics. Major functionality includes biomolecular database searching and retrieval, sequence and structure conservation analysis, ensemble normal mode analysis, protein structure and correlation network analysis, principal component, and related multivariate analysis methods. Here, we review recent package developments, including a new underlying segregation into separate packages for distinct analysis, and introduce a new method for structure analysis named ensemble difference distance matrix analysis (eDDM). The eDDM approach calculates and compares atomic distance matrices across large sets of homologous atomic structures to help identify the residue wise determinants underlying specific functional processes. An eDDM workflow is detailed along with an example application to a large protein family. As a new member of the Bio3D family, the Bio3D‐eddm package supports both experimental and theoretical simulation‐generated structures, is integrated with other methods for dissecting sequence‐structure–function relationships, and can be used in a highly automated and reproducible manner. Bio3D is distributed as an integrated set of platform independent open source R packages available from: http://thegrantlab.org/bio3d/.

Multidrug resistance is a serious problem in current chemotherapy. The efflux system largely responsible for resistance in Escherichia coli contains the drug transporter, AcrB. The structures of AcrB were solved in 2002 as the symmetric homo-trimer, and then in 2006 as the asymmetric homo-trimer. The latter suggested a functionally rotating mechanism. Here, by molecular simulations of the AcrB porter domain, we uncovered allosteric coupling and the drug export mechanism in the AcrB trimer. Allosteric coupling stabilized the asymmetric structure with one drug molecule bound, which validated the modelling. Drug dissociation caused a conformational change and stabilized the symmetric structure, providing a unified view of the structures reported in 2002 and 2006. A dynamic study suggested that, among the three potential driving processes, only protonation of the drug-bound protomer can drive the functional rotation and simultaneously export the drug.

G protein ␣ subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of G␣ i . Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.Heterotrimeric G proteins are key mediators of intracellular signaling pathways that control diverse cellular processes ranging from movement and division to differentiation and neuronal activity (1). G proteins consist of three subunits: G␣, G␤, and G␥. Bound with GDP, G␣ forms an inactive complex with its G␤␥ subunit partners. Interaction with activated receptor (GPCR) 3 promotes the exchange of GDP for GTP on G␣ and its separation from G␤␥. Both isolated G␣ and G␤␥ can then bind and activate or inhibit downstream effectors. GTP hydrolysis deactivates G␣, which subsequently reassociates with G␤␥ completing the cycle. This cycle is further regulated by two classes of additional proteins called regulators of G protein signaling. These function as either GTPase-activating proteins (which promote GTP hydrolysis) or GDP dissociation inhibitors (GDIs, which hinder exchange of GDP for GTP) (2). Important conformational transitions occurring at each stage of this regulated cycle have been characterized from extensive crystallographic studies. These include GDP, GTP analogue, G␤␥, GTPase-activating protein, GDI and most recently GPCR bound complex structures of G␣. However, the link between the observed conformations and the atomic level mechanisms involved in coupling receptor association, G protein activation, and effector interaction remain unclear.All G␣ proteins consist of a catalytic GTP binding Ras-like domain (termed RasD) and a heterotrimeric G protein specific helical domain (HD). Recent principal component analysis (PCA) of 53 available G␣ crystallographic structures identified three major conformationally distinct groups ( Fig. 1 and Ref. 3). These groups correspond to structures with bound GTP analogues, GDP, and GDI (red, green, and blue points in Fig. 1a, respectively). The major variation in the accumulated structures is the concerted displacements of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII), as well as a relatively small...