Unfolded DapA forms aggregates when diluted into free solution, confounding comparison with folding by the GroEL/GroES chaperonin system

Abstract

A recent hydrogen-deuterium exchange study of folding of the GroEL/GroES-dependent bacterial enzyme DapA has suggested that the DapA folding pathway when free in solution may differ from the folding pathway used in the presence of the GroEL/GroES chaperonin. Here, we have investigated whether DapA aggregation might be occurring in free solution under the conditions of the exchange experiment, as this would confound interpretation of the pathway predictions. Dynamic light scattering (DLS) data, sedimentation analysis and refolding yield indicate that significant aggregation occurs upon dilution of DapA from denaturant, bringing into question the earlier conclusion that different folding pathways occur in the absence and presence of the chaperonin system.

Keywords: Aggregation; DapA; GroEL; Light scattering; Protein folding.

Fig. 1. DapA aggregates upon dilution from denaturant directly into solution

DapA, denatured in guanidine HCl, was rapidly diluted into refolding buffer at 10°C, and this solution was pipetted into a cuvette equilibrated at 10°C in the sample compartment of a dynamic light scattering (DLS) instrument. Measurements were taken at 5 sec intervals for 5 min. The instrument software was used to estimate the size (hydrodynamic radius) of the scattering particles from the autocorrelation function at each acquisition point. These data are plotted vs. the acquisition time, starting at 5 sec after mixing. Data from two independent experiments are displayed (black and turquoise circles). In another experiment, denatured DapA was rapidly diluted (2.4 μM final concentration) into a solution containing 4.8 μM GroEL at 23°C. After 5 min, the solution was equilibrated at 10°C and placed in the DLS instrument. Measurements were taken and analyzed as for the dilution directly into solution (red squares). As controls, similar measurements were carried out with native DapA tetramer (24 μM; green triangles) and GroEL alone (4.8 μM; blue diamonds). Note that the estimated radii are averages calculated from the data at each acquisition time. The scattering particles in the direct dilution experiments were highly polydisperse, with average radii of 150-180 nm. Those in the GroEL-DapA complex experiment were somewhat polydisperse, and those in the control experiments were effectively monodisperse. Average hydrodynamic radii for these latter samples were: GroEL complex with DapA, 7.9 nm; GroEL, 6.8 nm; DapA tetramer, 3.3 nm.