Molecular Crowding Inhibits Intramolecular Breathing Motions in Proteins

Abstract

In aqueous solution some proteins undergo large-scale movements of secondary structures, subunits or domains -referred to as protein 'breathing' -that define a native-state ensemble of structures. These fluctuations are sensitive to the nature and concentration of solutes and other proteins and are thereby expected to be different in the crowded interior of a cell than in dilute solution. Here we use a combination of wide angle x-ray scattering (WAXS) and computational modeling to derive a quantitative measure of the spatial scale of conformational fluctuations in a protein solution. Concentration-dependent changes in the observed scattering intensities are consistent with a model of structural fluctuations in which secondary structures undergo rigid-body motions relative to one another. This motion increases with decreasing protein concentration or increasing temperature. Analysis of a set of five structurally and functionally diverse proteins reveals a diversity of kinetic behaviors. Proteins with multiple disulfide bonds exhibit little or no increase in breathing in dilute solutions. The spatial extent of structural fluctuations appears highly dependent on both protein structure and concentration and is universally suppressed at very high protein concentrations.

IgG Fab Fragments Forming Bivalent Complexes by a Conformational Mechanism That Is Reversible by Osmolytes

“…It has been reported that 50 -70% of proteins display some positive level of homodimerization (57), which brings up the possibility that the observations in the current work may reflect a more universal tendency among proteins. The phenomena of "protein breathing" and "folding funnels" (58,59) suggest that proteins can vacillate between a number of different conformations that represent entropically favorable free energy states. Perhaps certain of these spontaneous conformations create binding opportunity and entropic favorability for a bivalent Fab complex.…”

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

“…Such interactions, with a K d in the micromolar to millimolar range, are emerging as important components of the cell's signaling, regulatory, and stress adaptation mechanisms (6,8,9). Their transient nature is key to their function: Unlike tightly bound complexes, quinary interactions are highly sensitive to variations in their environment and respond rapidly to changes in temperature, pressure, pH, or the local concentration of surrounding molecules.…”

Detection of Functional Ligand-Binding Events Using Synchrotron X-Ray Scattering

“…The 2 peaks at q = 0.31 Å -1 and 0.60 Å -1 , as well as the trough at 0.50 Å -1 , are not only shifted in relative intensity upon ligand binding but in scattering angle as well (χ υ = 5.41). A calculation of the radius of gyration of apo-and (NAG) 3 -bound lysozyme from their atomic coordinates (1E8L and 1HEW, respectively) using the software CRYSOL 7 (version 2.4) confirms the implications of the scattering peak shifts, as upon (NAG) 3 binding, the Rg increases from 14.21 to 15.08.…”

“…Because the catalytic residues are located between subsites C and D, tetrasaccharide or longer oligosaccharides are enzymatically cleaved, whereas NAG, (NAG) 2 , and (NAG) 3 bind to subsites A, A/B, and A/B/C, respectively, acting as competitive inhibitors. Dissociation constants at pH 5.0 for NAG, (NAG) 2 , and (NAG) 3 decrease from 50 mM to 0.175 mM to 6.58 µM, a range of over 10 4 in magnitude. 13 This wide range offers the opportunity to assess the value of WAXS in the distinction of ligand-binding capacity between a series of structurally related small molecules.…”

“…13 This wide range offers the opportunity to assess the value of WAXS in the distinction of ligand-binding capacity between a series of structurally related small molecules. Solution WAXS patterns from hen egg white lysozyme were collected as previously described, 1,2 in the absence of ligand and the presence of dextrose, NAG, and (NAG) 3 . Figure 2a demonstrates the reproducibility of solution scattering over time (χ υ = 0.89).…”