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      Hidden alternate structures of proline isomerase essential for catalysis

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          Abstract

          A longstanding challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions 1, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations 1, 2. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a time scale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CypA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.

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          Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants

          W Kabsch (1993)
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            Protein dynamism and evolvability.

            The traditional view that proteins possess absolute functional specificity and a single, fixed structure conflicts with their marked ability to adapt and evolve new functions and structures. We consider an alternative, "avant-garde view" in which proteins are conformationally dynamic and exhibit functional promiscuity. We surmise that these properties are the foundation stones of protein evolvability; they facilitate the divergence of new functions within existing folds and the evolution of entirely new folds. Packing modes of proteins also affect their evolvability, and poorly packed, disordered, and conformationally diverse proteins may exhibit high evolvability. This dynamic view of protein structure, function, and evolvability is extrapolated to describe hypothetical scenarios for the evolution of the early proteins and future research directions in the area of protein dynamism and evolution.
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              Intrinsic dynamics of an enzyme underlies catalysis.

              A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                27 November 2009
                3 December 2009
                3 June 2010
                : 462
                : 7273
                : 669-673
                Affiliations
                [1 ]Department of Molecular and Cell Biology/QB3, University of California, Berkeley CA 94720-3220, USA
                [2 ]Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA
                Author notes
                Corresponding authors: (T.A.) tom@ 123456ucxray.berkeley.edu , 510-642-8758 (V), 510-666-2768 (FAX) and (D.K.) dkern@ 123456brandeis.edu , 781-736-2354 (V), 781-736-2316 (FAX)

                JSF, SD, and RE performed the X-ray experiments, MWC performed the NMR experiments, and MWC and JSF performed the activity and binding assays. JSF, MWC, DK, and TA analyzed data and wrote the paper.

                All authors contributed to data interpretation and commented on the manuscript.

                Article
                nihpa155226
                10.1038/nature08615
                2805857
                19956261
                9d714a83-1070-4d55-9d9b-35ea89574b5c

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM048958-16 ||GM
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