Lactose Repressor Experimental Folding Landscape: Fundamental Functional Unit and Tetramer Folding Mechanisms

The fundamental principles that govern monomer folding are believed to be congruent with those of protein oligomers. However, the effects of protein assembly during the folding reaction can result in a series of complex transitions that are considerably more challenging to deconvolute. Here we developed the experimental protein folding mechanism for the lactose repressor (LacI), for both the dimeric and the tetrameric states, using equilibrium unfolding and kinetic experiments, and by leveraging the previously reported monomer folding landscape. Reaction details for LacI oligomers were observed by way of circular dichroism, intrinsic fluorescence, and Förster resonance energy transfer (FRET) and as a function of protein concentration. In general, the dimer and tetramer are four-phase folding reactions in which the first three transitions are tantamount to the folding of constituent monomers. The final reaction phase of the LacI dimer can be attributed to protein assembly, based on the concentration dependence of the observed folding rates and intermolecular FRET measurements. Unlike the dimer, the latter reaction phase of the LacI tetramer is not dependent on protein concentration, likely because of a strong tethering of the monomers, which simplifies the folding reaction by eliminating an explicit protein assembly phase. Finally, folding of the LacI dimer and tetramer was assessed in the presence of polyethylene glycol to rule out inert molecular crowding as the driving force for the protein folding reaction; in addition, these data provide insight into the folding mechanism in vivo

Heat and mass transfer scale-up issues during freeze drying: II. Control and characterization of the degree of supercooling

This study aims to investigate the effect of the ice nucleation temperature on the primary drying process using an ice fog technique for temperature-controlled nucleation. In order to facilitate scale up of the freeze-drying process, this research seeks to find a correlation of the product resistance and the degree of supercooling with the specific surface area of the product. Freeze-drying experiments were performed using 5% wt/vol solutions of sucrose, dextran, hydroxyethyl starch (HES), and mannitol. Temperature-controlled nucleation was achieved using the ice fog technique where cold nitrogen gas was introduced into the chamber to form an “ice fog”, there-by facilitating nucleation of samples at the temperature of interest. Manometric temperature measurement (MTM) was used during primary drying to evaluate the product resistance as a function of cake thickness. Specific surface areas (SSA) of the freeze-dried cakes were determined. The ice fog technique was refined to successfully control the ice nucleation temperature of solutions within 1°C. A significant increase in product resistance was produced by a decrease in nucleation temperature. The SSA was found to increase with decreasing nucleation temperature, and the product resistance increased with increasing SSA. The ice fog technique can be refined into a viable method for nucleation temperature control. The SSA of the product correlates well with the degree of supercooling and with the resistance of the product to mass transfer (ie, flow of water vapor through the dry layer). Using this correlation and SSA measurements, one could predict scaleup drying differences and accordingly alter the freeze-drying process so as to bring about equivalence of product temperature history during lyophilization