Molecular crowding inhibits intramolecular breathing motions in proteins.
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.
Makowski
Lee
L
Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439-4845, USA. lmakowski@anl.gov
Rodi
Diane J
DJ
Mandava
Suneeta
S
Minh
David D L
DD
Gore
David B
DB
Fischetti
Robert F
RF
D000085:Acetates / Q000737:chemistry; D000818:Animals; D001360:Avidin / Q000737:chemistry; D002021:Buffers; D002417:Cattle; D002645:Chickens; D003198:Computer Simulation; D006454:Hemoglobins / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D009038:Motion; D009113:Muramidase / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D014961:X-Ray Diffraction / Q000379:methods
D000085:Acetates; D002021:Buffers; D006454:Hemoglobins; D009211:Myoglobin; D010710:Phosphates; D011506:Proteins; D001360:Avidin; D012710:Serum Albumin, Bovine; D009113:Muramidase