Evaluating the Consequences of Adsorption-Induced Structural Perturbations of Proteins

Abstract

Proteins usually fulfill their natural functions in the crowded cellular environment where they interact with a dense mixture of other biological macromolecules such as proteins. The concentration is very high, for example, up to several hundreds of mg/ml in cytosol of some E. coli, which is in contrast to the very dilute solutions used in most studies of function and structure. Here, we used Small-Angle Neutron Scattering (SANS) with the contrast matching technique to study the structure and oligomerization state of green fluorescent protein (GFP) in solutions containing various concentrations of the protein human serum albumin (HSA) as a crowding agent. By using HSA, a common protein in blood serum, we were creating a more biologically relevant crowding condition than other crowders usually used such as polyethylene glycol or Ficoll. GFP protein is a relatively stable protein with a beta-barrel structure, usually forming a dimer in dilute solution. By using perdeuterated GFP and hydrogenated HSA, we were able to probe only the GFP in the solutions by contrast matching HSA with an appropriate D2O/H2O buffer mixture. A series of HSA concentrations from 5mg/ml to 200 mg/ml were used. Analysis of the data indicates that GFP undergoes an HSA concentration-dependent transition that alters the way in which GFP oligomerizes in the solution. The dimer present in HSA-free solution remains unaltered by low concentrations of HSA. As the concentration of the crowder increases past 100 mg/mL, GFP adopts an ensemble of states with the transition to another dimeric configuration.