Klaus Gast

Fluoroalcohol-induced structural changes of proteins: some aspects of cosolvent-protein interactions

Abstract. The conformational transitions of bovine β-lactoglobulin A and phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have been studied by dynamic light scattering and circular dichroism spectroscopy in order to elucidate the potential of fluoroalcohols to bring about structural changes of proteins. Moreover, pure fluoroalcohol-water mixed solvents were investigated to prove the relation between cluster formation and the effects on proteins. The results demonstrate that cluster formation is mostly an accompanying phenomenon because important structural changes of the proteins occur well below the critical concentration of fluoroalcohol at which the formation of clusters sets in. According to our light scattering experiments, the remarkable potential of HFIP is a consequence of extensive preferential binding. Surprisingly, preferential binding seems to play a vanishing role in the case of TFE. However, the comparable Stokes radii of both proteins in the highly helical state induced by either HFIP or TFE point to a similar degree of solvation in both mixed solvents. This shows that direct binding or an indirect mechanism must be equally taken into consideration to explain the effects of alcohols on proteins. The existence of a compact helical intermediate with non-native secondary structure on the transition of β-lactoglobulin A from the native to the highly helical state is clearly demonstrated.

Effect of environmental conditions on aggregation and fibril formation of barstar

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross-β structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75xa0mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-state→fibril type I→fibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.

Reduced-denatured ribonuclease A is not in a compact state

Dynamic light scattering and circular dichroism experiments were performed to determine the compactness and residual secondary structure of reduced and by 6 M guanidine hydrochloride denatured ribonuclease A. We find that reduction of the four disulphide bonds by dithiothreitol at 20°C leads to total unfolding and that a temperature increase has no further effect on the dimension. The Stokes radius of ribonuclease A at 20°C is R s = (1.90 ± 0.04) nm (native) and R s = (3.14 ± 0.06) nm (reduced‐denatured). Furthermore, circular dichroism spectra do not indicate any residual secondary structure. We suggest that reduced‐denatured Ribonuclease A has a random coil‐like conformation and is not in a compact denatured state.

Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Abstract Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 °C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available.

Initial hydrophobic collapse is not necessary for folding RNase A

BACKGROUNDnOne of the main distinctions between different theories describing protein folding is the predicted sequence of secondary structure formation and compaction during the folding process. Whether secondary structure formation precedes compaction of the protein molecules or secondary structure formation is driven by a hydrophobic collapse cannot be decided unequivocally on the basis of existing experimental data.nnnRESULTSnIn this study, we investigate the refolding of chemically denatured, disulfide-intact ribonuclease A (RNase A) by monitoring compaction and secondary structure formation using stopped-flow dynamic light scattering and stopped-flow CD, respectively. Our data reveal the formation of a considerable amount of secondary structure early in the refolding of the slow folding species of RNase A without a significant compaction of the molecules. A simultaneous formation of secondary structure and compaction is observed in the subsequent rate-limiting step of folding.nnnCONCLUSIONSnDuring folding of RNase A an initial global hydrophobicity is not observed, which contradicts the view that this is a general requirement for protein folding. This folding behavior could be typical of similar, moderately hydrophobic proteins.

Cold denaturation of yeast phosphoglycerate kinase: which domain is more stable?

Under destabilising conditions both heat and cold denaturation of yeast phosphoglycerate kinase (PGK) can be observed. According to previous interpretation of experimental data and theoretical calculations, the C‐terminal domain should be more stable than the N‐terminal domain at all temperatures. We report on thermal unfolding experiments with PGK and its isolated domains, which give rise to a revision of this view. While the C‐terminal domain is indeed the more stable one on heating, it reveals lower stability in the cold. These findings are of importance, because PGK has been frequently used as a model for protein folding and mutual domain interactions.

Ribonuclease T1 has different dimensions in the thermally and chemically denatured states: a dynamic light scattering study.

© 1997 Federation of European Biochemical Societies.

CONFORMATION OF THERMALLY DENATURED RNASE T1 WITH INTACT DISULFIDE BONDS :A STUDY BY SMALL-ANGLE X-RAY SCATTERING

Small-angle X-ray scattering of RNase T1 with intact disulfide bonds was measured at 20 degrees and 60 degrees C in order to get insight into the structural changes of the protein caused by thermal denaturation. The radius of gyration increases from R(G)= 1.43 nm to R(G) = 2.21 nm. The conformations of the molecules at 60 degrees C are similar to those of ring-shaped random walk chains. However, the molecules are more compact than one would expect under theta conditions due to attractive interactions between the chain segments. The volume needed for free rotation of the thermally unfolded protein molecules about any axis in solution is five times greater than in the native state whereas the hydrodynamic effective volume is increasing only two times.

Time-resolved dynamic light scattering as a method to monitor compaction during protein folding

The mechanisms and the dynamics of protein folding are subject of a still increasing number of theoretical and experimental studies. While spectroscopic methods are already used for many years to measure the folding rates and to monitor the formation of secondary and tertiary structure, kinetic measurements of the compactness are only beginning to emerge. Time-resolved dynamic light scattering (DLS) is a useful tool to follow the compaction during protein folding by measuring the hydrodynamic Stokes radius RS. Additionally, changes in the state of association can be detected by simultaneous measurements of the scattering intensity. The usefulness of different techniques for time-resolved DLS measurements and the general limits for kinetic DLS experiments are discussed first. Then we describe the adaptation of a stopped-flow system (SFM-3) to a DLS apparatus, the particular data acquisition schemes, and the experimentally attainable limits. The feasibility of stopped-flow DLS is demonstrated by the results of folding investigations with ribonuclease A, phosphoglycerate kinase, and bovine α-lactalbumin. Refolding was initiated by denaturant dilution jumps, which were repeated up to 100 times in order to obtain a reasonable signal-to-noise ratio. Kinetic DLS experiments can be performed fairly with a time resolution of one second. The time resolution of 100ms is probably the attainable limit. The capabilities of time-resolved DLS and time-resolved small-angle X-ray scattering are compared.