Essential Domain Motions in Barnase Revealed by MD Simulations Svetlana B. Nolde, 1 Alexander S. Arseniev, 1 Vladislav Yu Orekhov, 2 and Martin Billeter 3 * 1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry (RAS), Moscow, Russia 2 Swedish NMR Centre at Go ¨ teborg University, Go ¨ teborg, Sweden 3 Biochemistry and Biophysics, Go ¨ teborg University, Go ¨ teborg, Sweden ABSTRACT The wealth of data accumulated on the bacterial ribonuclease barnase is comple- mented by molecular dynamics trajectories starting from four different experimental structures and covering a total of >10 ns. Using principal compo- nent analysis, the simulations are interpreted in view of dynamic domains and hinges promoting relative motions of these domains. Two domains with residues 7–22 and 52–108 for the first domain and residues 25–51 for the second domain were consistently observed. Hinge regions consist primar- ily of Tyr24, Ser50, Ile51, and Gly52. Earlier muta- tion studies have demonstrated that the residues of the hinge regions play essential roles for the stabil- ity and activity of barnase. The domain motions are correlated to inter-domain interactions involving functionally important active site residues, such as Lys27 and Glu73. A model is presented that com- bines the observation of dynamic domains and their motions with the extensive mutation data from the literature. Enthalpic energy contributions originat- ing from specific inter-domain interactions as well as entropic energy contributions due to the domain motions are discussed in the frame of this model and compared with destabilization energies measured for corresponding mutants. Proteins 2002;46:250 –258. © 2002 Wiley-Liss, Inc. Key words: dynamic domains; entropy; essential dy- namics; hinge bending; molecular dy- namics simulation; principal compo- nent analysis; protein activity; protein stability INTRODUCTION The highly complex character of proteins allows these macromolecules to adopt a multitude of different folds and thus to support a large variety of specific functions. For a deeper understanding of the behavior exhibited by a protein, knowledge of its structure is not sufficient; charac- terization of the internal dynamics and the identification of functionally relevant amino acid residues are required as well. The description of the static structure of a protein, including all its side-chains, is complicated by the neces- sity to consider the dynamic behavior, or more generally entropic contributions, to explain protein stability and activity. Many efforts in protein engineering start from characterizations and comparisons of structures, an ap- proach that has been termed “structure-led.” 1 An alterna- tive, “function-led,” starting point consists of biological screening procedures of protein variants, typically ob- tained by single site mutations, followed by a scoring of the change in stability and/or activity. 1 The description of relations between structure and function requires a map- ping of the localized mutations obtained with the latter approach to the more global features of structure and internal dynamics. However, a simple localization of func- tion-relevant mutations on a rigid structure often fails in providing consistent structure–function relationships. More promising is a view combining structural and dynamic data, the latter preferably at various time scales, as well as screening results on stability, activity, and specificity. Extensive studies on the bacterial ribonuclease barnase were initiated during the 1960s, when this protein was suggested as an ideal model for protein folding. 2,3 Since that time, a wealth of data has been collected for barnase. Experimental and computational investigations of folding pathways and kinetics of barnase were combined with detailed analyses of its structure and function, and with protein engineering experiments (see, e.g., refs. 4 and 5). Hundreds of mutations have been introduced covering all 110 positions of the barnase sequence. 6,7 Numerous crys- tallographic and nuclear magnetic resonance (NMR) stud- ies provide high-resolution structures of barnase in vari- ous forms. The Protein Data Bank (PDB) 8 contains more than 35 entries with barnase, such as wild-type and mutated forms and complexes with inhibitors. NMR inves- tigations have complemented these structural characteriza- tions with descriptions of the internal dynamics of the molecule. 9,10 In an attempt to combine the reliability of experimental NMR data on structure and dynamics with the higher resolution in space and time achieved by computer simulation of the molecular dynamics (MD), we Abbreviations: MD, molecular dynamics; NMR, nuclear magnetic resonance; PCA, principal component analysis; PDB, Protein Data Bank; RMSD, root-mean-square deviation. Grant sponsor: Swedish Science Foundation NFR; Grant number: K-AA/KU 12071-300; Grant number: K-AA/KU 12071-303; Grant sponsor: Russian Foundation for Basic Research; Grant number: 99-04-48834; Grant number: 00-04-55024. *Correspondence to: Martin Billeter, Biochemistry and Biophysics, Go ¨ teborg University, Box 462, 40530 Go ¨teborg, Sweden. E-mail: martin.billeter@bcbp.gu.se Received 5 April 2001; Accepted 7 September 2001 Published online 00 Month 2001 PROTEINS: Structure, Function, and Genetics 46:250 –258 (2002) DOI 10.1002/prot.10030 © 2002 WILEY-LISS, INC.