Probing the structure and stability of a hybrid protein: the human-E. coli thioredoxin chimera.

The structure and stability of a hybrid protein composed of N-terminal human and C-terminal E. coli thioredoxin domains were investigated by NMR, fluorescence, and circular dichroism spectroscopy. We demonstrate that the chimeric protein is correctly folded and exhibits the common thioredoxin architecture. However, the stability of the hybrid protein toward thermal and chemical denaturation is clearly reduced when compared with both parent proteins. Altogether, our data indicate that the interface between the two folding units of thioredoxin is tolerant toward changes in exact interdigitation of side chains, allowing for the formation of the unique overall thioredoxin fold. Further, the gene encoding the human-E. coli chimera was tested in vivo whether it supports the assembly of filamentous phages. No complementation of a thioredoxin-deficient E. coli mutant for the replication of the phages M13 or fd was observed, suggesting that parts of the overall protein structure in the N-terminal domain are crucial for this activity.

Denatured collapsed states in protein folding: example of apomyoglobin.

Experimental approaches, including circular dichroism, small angle X-ray scattering, steady-state fluorescence, and fluorescence energy transfer, were applied to study the 3D-structure of apomyolgobin in different conformational states. These included the native and molten globules, along with either less ordered conformations induced by the addition of anions or completely unfolded states. The results show that the partially folded forms of apomyoglobin stabilized by KCl and/or Na(2)SO(4) under unfolding conditions (pH 2) exhibit a significant amount of secondary structure (circular dichroism), low packing density of protein molecules (SAXS), and native-like dimensions of the AGH core (fluorescence energy transfer). This finding indicates that a native-like tertiary fold of the polypeptide chain, i.e., the spatial organization of secondary structure elements, most likely emerges prior to the formation of the molten globule state.

Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1.

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.

Multisite fluorescence in proteins with multiple tryptophan residues. Apomyoglobin natural variants and site-directed mutants.

Time-resolved fluorescence experiments were carried out on a variety of apomyoglobins with one or two tryptophan (Trp) residues located at invariant positions 7 and 14 in the primary sequence. In all cases, the Trp fluorescence kinetics were resolved adequately into two discrete lifetime domains, and decay-associated spectra (DAS) were obtained for each decay component. The DAS resolved for unfolded proteins were indistinguishable by position of the emission maxima and the spectral shapes. The folded proteins revealed noticeable differences in the DAS, which relate to the diverse local environments around the Trp residues in the individual proteins. Furthermore, the DAS of wild-type protein possessing two Trp residues were simulated well by that of one Trp mutants either in the native, molten globule, or unfolded states. Overall, employing Trp fluorescence and site-directed mutagenesis allowed us to highlight the conformational changes induced by the single amino acid replacement and generate novel structural information on equilibrium folding intermediates. Specifically, it was found that conformational fluctuations in the local cluster around the evolutionarily conserved Trp(14) are very similar in the native and molten globule states of apomyoglobins. This result indicates that residues in the E and B helices contributing to this cluster are most likely involved in the stabilization of the overall architecture of the structured molten globule intermediate.

Nanosecond dynamics of tryptophans in different conformational states of apomyoglobin proteins.

Fluorescence anisotropy kinetics were employed to quantify the nanosecond mobility of tryptophan residues in different conformational states (native, molten globule, unfolded) of apomyoglobins. Of particular interest is the similarity between the fluorescence anisotropy decays of tryptophans in the native and molten globule states. We find that, in these compact states, tryptophan residues rotate rapidly within a cone of semiangle 22-25 degrees and a correlation time of 0.5 ns, in addition to rotating together with the whole protein with a correlation time of 7-11 ns. The similar nanosecond dynamics of tryptophan residues in both states suggests that the conformation changes that distinguish the molten globule and native states of apomyoglobins originate from either subtle, slow rearrangements or fast changes distant from these tryptophans.

Molten globule versus variety of intermediates: influence of anions on pH-denatured apomyoglobin.

The molten globule state was shown to be the third thermodynamic state of protein molecules in addition to their native and unfolded states. On the other hand, it was reported that optical and hydrodynamic properties of pH-denatured apomyoglobin depend on the nature of anions added to the protein solution. This observation was used to conclude that there are many 'partly folded' intermediates between the native and unfolded states rather than one distinct molten globule state. However, little is known on the structures of pH-denatured apomyoglobin in the presence of different anions. Two tyrosine residues in horse apomyoglobin have been successively modified by the reaction with tetranitromethane. This approach was employed to measure the distances between tryptophans and modified tyrosines in different states of apomyoglobin by the method of direct energy transfer. Experimental data show that the distance between the middle of the A-helix and the beginning of the G-helix and/or the end of the H-helix in 'anion-induced' states are very close to those in the native holo- and apomyoglobins. This suggests that the AGH helical complex, being the most structured part of apomyoglobin in the molten globule state, exists also in pH-denatured apomyoglobin in the presence of different anions. Consequently, all non-native forms of apomyoglobin studied so far share the common important feature of its native structure.

Direct energy transfer to study the 3D structure of non-native proteins: AGH complex in molten globule state of apomyoglobin.

The direct energy transfer technique was modified and applied to probe the relative localization of apomyoglobin A-, G- and H-helixes, which are partly protected from deuterium exchange in the equilibrium molten globule state and in the molten globule-like kinetic intermediate. The non-radiative transfer of tryptophan electronic energy to 3-nitrotyrosine was studied in different conformational states of apomyoglobin (native, molten globule, unfolded) and interpreted in terms of average distances between groups of the protein chain. The experimental data show that the distance between the middle of A-helix and the N-terminus of G-helix as well as the distance between the middle of the A-helix and the C-terminus of the H-helix in the molten globule state are close to those in the native state. This is a strong argument in favor of similarity of the overall architecture of the molten globule and native states.