Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A.

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.

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.

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

Ribonuclease T1 can be unfolded and refolded without forming noticeable amounts of aggregates allowing to characterise the dimensions of a protein in different denatured states in terms of the Stokes radius RS. Upon thermal unfolding RS increases from 1.74 nm at 20 degrees C to 2.14 nm at 60 degrees C. By contrast, RS = 2.40 nm was obtained at 5.3 M guanidinium chloride (GuHCl) and 20 degrees C. Heating from 20 degrees C to 70 degrees C in the presence of 5.3 M GuHCl led to a 5% decrease in RS.

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 degrees 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 degrees 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.

Prothymosin alpha: a biologically active protein with random coil conformation.

Prothymosin is an acidic protein with an unusual amino acid composition. Though its exact function is not yet known, its high evolutionary conservation and wide tissue distribution suggest an essential biological role. Its physical state, which is controversially discussed in previous publications, was investigated using small-angle X-ray scattering, dynamic light scattering, mass spectrometry, and circular dichroism (CD). Our results unequivocally demonstrate that prothymosin is a monomer under physiological conditions. The protein adopts a random coillike conformation but exhibits persistence of direction and curvature. No regular secondary structure is detectable by CD. The Stokes radius, Rs = 3.07 nm, and the radius of gyration, RG = 4.76 nm, are 1.77 and 3.42 times larger, respectively, than those expected for a compactly folded protein consisting of 109 amino acid residues. A remarkable amount of secondary structure is formed only in the presence of trifluoroethanol at low pH. The finding that a biologically active protein molecule with 109 amino acid residues adopts a random coil conformation under physiological conditions raises the question whether this is a rare or a hitherto-overlooked but widespread phenomenon in the field of macromolecular polypeptides.

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.

Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate.

Apomyoglobin undergoes a two-step unfolding transition when the pH is lowered from 6 to 2. The partly folded intermediate (I) state at pH 4 and low ionic strength has properties of a molten globule. We have studied structural features of this state, its compactness, content of secondary structure, and specific packing of aromatic side chains, using dynamic light scattering, and small-angle X-ray scattering and far- and near-ultraviolet circular dichroism spectroscopy. Particular attention was paid to temperature-dependent structural changes. The results are discussed with reference to the native-like (N) state and the highly unfolded (U) state. It turned out that the I-state is most compact near 30 degrees C, having a Stokes radius 20% larger and a radius of gyration 30% larger than those of the N-state. Both cooling and heating relative to 30 degrees C led to an expansion of the molecule, but the structural changes at low and high temperatures were of a different kind. At temperatures above 40 degrees C non co-operative melting of structural elements was observed, while the secondary structure was essentially retained on cooling. The results are discussed in context with theoretical predictions of the compactness and the stability of apomyoglobin by Alonso et al. [Alonso, D. O. V., Dill, K. A., and Stigter, D. (1991) Biopolymers 31:1631-1649]. Comparing the I-state of apomyoglobin with the molten globules of alpha-lactalbumin and cytochrome c, we found that the compactness of the molten globule states of the three proteins decreases in the order alpha-lactalbumin > apocytochrome c > apomyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450.

The steroid 11ß-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4-5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011ß system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011α from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11ß-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M(r) 60 kD) and a NADPH-cytochrome P450 reductase (M(r) 79 kD).