The role of unstructured extensions in the rotational diffusion properties of a globular protein: the example of the titin i27 module.
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.
Centro Interfacoltà Misure, University of Parma, Parma, Italy.
Giuseppe
G
Nicastro
Paola
P
Margiocco
Barbara
B
Cardinali
Paola
P
Stagnaro
Fabio
F
Cauglia
Carla
C
Cuniberti
Maddalena
M
Collini
David
D
Thomas
Annalisa
A
Pastore
Mattia
M
Rocco
D000465:Algorithms; D003198:Computer Simulation; D064211:Connectin; D004058:Diffusion; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry*; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012399:Rotation; D013050:Spectrometry, Fluorescence
D064211:Connectin; D009124:Muscle Proteins; D009587:Nitrogen Isotopes; D011494:Protein Kinases