[Y97V substitution in the horse cytochrome c causes accumulation of the equilibrium intermediate]. / Zamena Y97V v tsitokhrome c loshadi privodit k nakopleniiu ravnovesnogo intermediata.

Equilibrium unfolding experiments on several mutant forms of horse heart cytochrome c were performed. By means of absorbance spectroscopy, the accumulation of an equilibrium intermediate was revealed upon unfolding of Y97V mutant protein, and its structural properties were characterized. The data obtained allow one to conclude that the equilibrium intermediate corresponds to the earliest kinetic intermediate Ic in cytochrome c folding reaction. A comparative analysis of spectral properties of unfolded states of cytochrome c induced by urea or guanidine hydrochloride is presented.

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.

Non-functional conserved residues in globins and their possible role as a folding nucleus.

Structure-based sequence alignment of 728 sequences of different globin subfamilies shows that in each subfamily there are two clusters of consensually conserved residues. The first is the well-known "functional" cluster which includes six heme-binding conserved residues (Phe CD1, His F8; aliphatic E11, FG5; hydrophobic F4, G5) and seven other conserved residues (Pro C2; aliphatic H19; hydrophobic B10, B13, B14, CD4, E4) that do not bind the heme but belong to its immediate neighborhood. The second cluster revealed here (aliphatic A8, G16, G12; aromatic A12; hydrophobic H8 and possibly H12) is distant from the heme. It is entirely non-polar and includes one turn (i, i+4 positions) from each of helices A, G, and H. It is known that A, G, and H helices formed at the earliest stage of apomyoglobin folding remain relatively stable in the equilibrium molten globule state, and are likely to be tightly packed with each other in this state. We have shown the existence of two similar conserved clusters in c -type cytochromes, heme-binding and distal from the heme. The second cluster in c -cytochromes includes one turn from each of the N and C-terminal alpha-helices. These N and C-terminal helices in cytochrome c are formed at the earliest stage of protein folding, remain relatively stable in the molten globule state, and are tightly packed with each other in this state, similar to the observed behavior of the globins. At least these two large protein families (c -type cytochromes and globins) have a close similarity in the existence and mutual positions of non-functional conserved residues. We assume that non-functional conserved residues are requisite for the fast and correct folding of both of these protein families into their stable 3D structures.

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.

Protein evolution and protein folding: non-functional conserved residues and their probable role.

It is shown that there are two types of conserved residues in evolutionary and functionally related proteins whose sequences have been well diverged in evolution. The first group consists of residues forming the active center, while the second (first established in this work) has nothing to do with function and therefore should be related to protein structure and/or protein folding. The lattter group consists of 4 residues in c-type cytochromes and 6 residues in globins. All these residues belong to alpha-helices and occupy positions (i, i + 4) or (i, i + 3), stabilizing one helical turn in some helices. These residues form an interface between the N- and C-terminal helices in c-type cytochromes and helices A, G, H in globins. These helical complexes form early in protein folding and are relatively stable in both equilibrium and kinetic folding intermediates. The attractive hypothesis is that these helices form folding nuclei in protein in the frame of the nucleation-growth mechanism of protein folding.

Empirical solvent-mediated potentials hold for both intra-molecular and inter-molecular inter-residue interactions.

Whether knowledge-based intra-molecular inter-residue potentials are valid to represent inter-molecular interactions taking place at protein-protein interfaces has been questioned in several studies. Differences in the chain connectivity effect and in residue packing geometry between interfaces and single chain monomers have been pointed out as possible sources of distinct energetics for the two cases. In the present study, the interfacial regions of protein-protein complexes are examined to extract inter-molecular inter-residue potentials, using the same statistical methods as those previously adopted for intra-molecular residue pairs. Two sets of energy parameters are derived, corresponding to solvent-mediation and "average residue" mediation. The former set is shown to be highly correlated (correlation coefficient 0.89) with that previously obtained for inter-residue interactions within single chain monomers, while the latter exhibits a weaker correlation (0.69) with its intra-molecular counterpart. In addition to the close similarity of intra- and inter-molecular solvent-mediated potentials, they are shown to be significantly more residue-specific and thereby discriminative compared to the residue-mediated ones, indicating that solvent-mediation plays a major role in controlling the effective inter-residue interactions, either at interfaces, or within single monomers. Based on this observation, a reduced set of energy parameters comprising 20 one-body and 3 two-body terms is proposed (as opposed to the 20 x 20 tables of inter-residue potentials), which reproduces the conventional 20 x 20 tables with a correlation coefficient of 0.99.

Release of retinol and denaturation of its plasma carrier, retinol-binding protein.

BACKGROUND: Retinol is tightly packed inside the structure of its plasma carrier (retinol-binding protein, RBP). It was found that retinol release from RBP to aqueous solutions is facilitated by either very low pH or very high temperatures (i.e. by non-physiological conditions that cause protein denaturation). It was also found that alcohols induce protein conformational transitions to denatured states. On this basis, it may be suggested that retinol release in vivo is facilitated by the partial unfolding of the carrier resulting from the concerted action of the moderate local decrease of pH and the moderate local decrease of dielectric constant in proximity to the target membranes. RESULTS: In vitro, at 37 degrees C, retinol is removed from its plasma carrier by the concerted action of the moderately low pH and the moderately low dielectric constant of solutions containing a low ionic strength buffer and methanol in variable proportions. Release of retinol is accompanied by a conformational transition of RBP from the native to the molten-globule state. CONCLUSIONS: The physiological function of RBP-targeted delivery of retinol-is mimicked in vitro by the facilitated release of retinol (associated with a partial unfolding of the protein carrier) in solutions exhibiting pH and dielectric constant values that are within the range of values expected in the in vivo microenvironment.

[Effect of a biologically active interferon fragment on the structure of the synthetic protein carrier]. / Issledovanie vliianiia biologicheski aktivnogo fragmenta interferona na strukturu iskusstvennogo belka-nositelia.

A biologically active de novo protein albeferon was studied by physical techniques including CD spectroscopy in far and near ultraviolet regions and microcalorimetry. Albeferon was obtained by grafting an active octapeptide interferon fragment into a de novo protein albebetin used as a carrier. It was shown that attachment of the octapeptide into its molecule did not weaken albebetin but even slightly improved its structure and stability. The obtained results can be used for de novo protein design and improvement.