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Biophysical Journal 87:1227-1240 (2004)
© 2004 The Biophysical Society

The Role of Unstructured Extensions in the Rotational Diffusion Properties of a Globular Protein: The Example of the Titin I27 Module

Giuseppe Nicastro * {dagger}, Paola Margiocco {ddagger}, Barbara Cardinali {ddagger} §, Paola Stagnaro {ddagger}, Fabio Cauglia §, Carla Cuniberti §, Maddalena Collini ¶, David Thomas ||, Annalisa Pastore {dagger} and Mattia Rocco {ddagger}

* Centro Interfacoltà Misure, University of Parma, Parma, Italy; {dagger} National Institute for Medical Research, The Ridgeway, London NW71AA, United Kingdom; {ddagger} S.C. Medicina Rigenerativa, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; § Dipartimento di Chimica e Chimica Industriale, Università di Genova, Genoa, Italy; Dipartimento di Fisica, Università di Milano Bicocca and INFM, Milan, Italy; and || Biological NMR Unit, School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

Correspondence: Address reprint requests to Annalisa Pastore, E-mail: apastor{at}nimr.mrc.ac.uk.

The possibility of predicting the overall shape of a macromolecule in solution from its diffusional properties has gained increasing importance in the structural genomic era. Here we explore and quantify the influence that unstructured and flexible regions have on the motions of a globular protein, a situation that can occur from the presence of such regions in the natural sequence or from additional tags. I27, an immunoglobulin-like module from the muscle protein titin, whose structure and properties are well characterized, was selected for our studies. The backbone dynamics and the overall tumbling of three different constructs of I27 were investigated using 15N NMR relaxation collected at two 15N frequencies (60.8 and 81.1 MHz) and fluorescence depolarization spectroscopy after labeling of a reactive cysteine with an extrinsic fluorophore. Our data show that the presence of disordered tags clearly exerts a frictional drag that increases with the length of the tags, thus affecting the module tumbling in solution. We discuss the use and the limitations of current approaches to hydrodynamic calculations, especially when having to take into account local flexibility.




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