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

Abstract

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 (15)N NMR relaxation collected at two (15)N 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.

Selective observation of the disordered import signal of a globular protein by in‐cell NMR: The example of frataxins

“…Additionally, while CyaY is globular and roughly isotropic, Yfh1 contains an unfolded tail which greatly increases the anisotropy of the protein. It was previously demonstrated that an unstructured tag of 11 residues increases of about 30% the correlation time of the titin domain I27 . Our results thus exclude the formation of appreciable quantities of large aggregates since the observed values would barely account for a dimer.…”

NMR data collection and analysis protocol for high-throughput protein structure determination

“…b Protein precipitated during data collection with a rate of 6% per day. c Molecular masses are listed for U-[ 15 N, 13 C] labeled protein with His-tag [since tags affect r (38)]͞for expected in vivo expressed protein. d Recorded with cryogenic probe at 600 MHz.…”

Ensemble Structure of the Modular and Flexible Full-Length Vesicular Stomatitis Virus Phosphoprotein

“…2f and g). The presence of long IDRs in VSV P exerts a frictional drag that slows down the overall tumbling rate of the molecule 62,63 and may explain the decrease of signal intensity observed for resonances in the globular domains as compared to the spectra of the isolated domains ( Fig. 2c and d).…”