Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Abstract

Small-angle neutron scattering was used to study the effects of macromolecular crowding by two globular proteins, i.e., bovine pancreatic trypsin inhibitor and equine metmyoglobin, on the conformational ensemble of an intrinsically disordered protein, the N protein of bacteriophage λ. The λ N protein was uniformly labeled with (2)H, and the concentrations of D2O in the samples were adjusted to match the neutron scattering contrast of the unlabeled crowding proteins, thereby masking their contribution to the scattering profiles. Scattering from the deuterated λ N was recorded for samples containing up to 0.12 g/mL bovine pancreatic trypsin inhibitor or 0.2 g/mL metmyoglobin. The radius of gyration of the uncrowded protein was estimated to be 30 Å and was found to be remarkably insensitive to the presence of crowders, varying by <2 Å for the highest crowder concentrations. The scattering profiles were also used to estimate the fractal dimension of λ N, which was found to be ∼1.8 in the absence or presence of crowders, indicative of a well-solvated and expanded random coil under all of the conditions examined. These results are contrary to the predictions of theoretical treatments and previous experimental studies demonstrating compaction of unfolded proteins by crowding with polymers such as dextran and Ficoll. A computational simulation suggests that some previous treatments may have overestimated the effective volumes of disordered proteins and the variation of these volumes within an ensemble. The apparent insensitivity of λ N to crowding may also be due in part to weak attractive interactions with the crowding proteins, which may compensate for the effects of steric exclusion.

Equilibrium simulation of trp-cage in the presence of protein crowders

“…[1][2][3][4][5] It is well established that excluded-volume interactions with crowder particles can have a significant stabilizing effect on globular proteins, as demonstrated by experiments with synthetic crowding agents such as Ficoll or dextran. 6 Today, experiments are increasingly often performed under more realistic crowding conditions, using concentrated protein solutions [7][8][9][10] or cells. 9,[11][12][13][14][15][16] In these systems, it has been shown that other interactions may dominate over the steric repulsions and lead to a net destabilization of globular proteins.…”

“…18,19 As crowding agent, we pick the 58-residue bovine pancreatic trypsin inhibitor (BPTI), which was used in recent experiments on the disordered N protein of bacteriophage λ. 10 We model the BPTI molecules in full atomistic detail but assume them to stay folded with restricted internal dynamics. For comparison, we also perform simulations with simple hard-sphere crowders.…”

Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo

“…The physiological role of the COR15 proteins is to stabilize chloroplast membranes at mild freezing temperatures between approximately 25°C and 215°C, at which extracellular ice crystallization leads to partial cell dehydration and increased molecular crowding. Macromolecular crowding, applied either through high concentrations of globular proteins or of polymers such as dextran, has generally only limited effects on IDP structure (Mouillon et al, 2008;Szasz et al, 2011;Goldenberg and Argyle, 2014). However, osmolyte-induced crowding was also ineffective to induce folding in some plant LEA proteins (Mouillon et al, 2008), although it can induce refolding of denatured globular proteins (Davis-Searles et al, 2001).…”

Effects of Macromolecular Crowding on the Collapse of Biopolymers

“…The specific value of x c should in principle vary with the nature of interactions in the ternary system of polymer, crowding particles, solvent, and N . Nevertheless, the estimated x c is a guide to obtain an approximate estimate of φ c , and we show below [34], and λN in BPTI or metmyoglobin (Mb) [35]. To compare with experiments, we superimposed our simulation results with N = 100 and λ = 0.9, 1.9, 3.8.…”