Combined Use of NMR Relaxation Measurements and Hydrodynamic Calculations To Study Protein Association. Evidence for Tetramers of Low Molecular Weight Protein Tyrosine Phosphatase in Solution

Abstract

We describe a novel method for determining weak association constants of oligomeric protein complexes formed transiently under equilibrium conditions. This type of equilibrium process is recognized as being biologically important, but generally hard to study. Heteronuclear spin relaxation rates measured at multiple protein concentrations are analyzed using relaxation rates predicted from hydrodynamic calculations, yielding equilibrium constants and structural characterization of the protein complexes. The method was used to study the oligomerization equilibrium of bovine low molecular weight protein tyrosine phosphatase. X-ray structures of monomeric and dimeric forms of the protein have been reported previously. Using longitudinal and transverse (15)N relaxation rates measured at four different protein concentrations, we detected the monomer, dimer, and a previously unknown tetramer and measured the dissociation constants of the equilibria involving these species. A comparison of experimental and predicted relaxation rates for individual backbone amide (15)N spins enabled delineation of the tetramerization interface. The results suggest a novel concept for substrate modulation of enzymatic activity based on a "supramolecular proenzyme". The fast and reversible switching of the "supramolecular proenzyme" would have obvious advantages for the regulation of enzymes involved in cell signaling pathways.

NMR Reveals a Novel Glutaredoxin–Glutaredoxin Interaction Interface

“…60 The shell modelling approach is characterized by a single parameter a, the atomic element radius that represents the sum of the thickness of the hydration shell and the average van der Waals radius in the molecule. Nevertheless, the choice of the a parameter is a critical point and needs to be optimized against the experimental relaxation data.…”

Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from 15N relaxation data using computer program ROTDIF

“…In particular, a failure to take the rotational anisotropy into account could result in spurious conformational exchange motions. Other emerging applications include structure characterization in multidomain systems [5][6][7][8] and analysis of protein-ligand interactions [5,9] and protein association [10].…”

Quantitative Analysis of Biomolecular NMR Spectra: A Prerequisite for the Determination of the Structure and Dynamics of Biomolecules

“…The prediction of T1/T2 values in the frame of HYDRONMR [264] was proposed to be used to distinguish the residues undergoing chemical exchange from those undergoing diffusion anisotropy. The combined use of NMR relaxation measurements and hydrodynamic calculations was used [265] to study protein oligomerisation. Similarly, protein selfdiffusion was studied in parallel with 15 N relaxation [266].…”