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The interaction of apomyoglobin and its mutant forms with phospholipid membranes was studied using tryptophan fluorescence and CD in the far UV-region. It is shown that a negatively charged phospholipid membrane can have a double effect on the structure of protein molecule upon their interaction: it denatures the native structure of the protein to its intermediate state similar to that in solution, acting as a moderately denaturing reagent. On the other hand, it can structure the unfolded protein to the same intermediate state stabilizing its structure. The kinetics of interaction between the protein and its mutant forms and the phospholipid membrane depends on the charge of the membrane surface. Here the rate of this interaction depends on the phospholipids vesicle concentration and the protein molecule stability increasing with a decrease of the latter. The importance of the obtained results for the folding of membrane proteins and the choice of the pathway for target delivery of protein drugs are discussed.

The effect of some amino acid residues in A, B, G, and H helices on the folding nucleus and folding intermediate state formation was estimated. For four apomyoglobin mutant forms with point replacements of hydrophobic amino acid residues by Ala, the influence of the substitutions on the stability of native (N) protein and its folding intermediate state (I) was studied, as well as on the protein folding/unfolding rates. Equilibrium and kinetic studies on mutant proteins over a wide range of urea concentrations have shown that the protein native state was strongly destabilized in comparison with that of the wild type protein. At the same time, stability of the intermediate state changed insignificantly. It was shown that amino acid residues of A, G, and H helices make a small contribution to apomyoglobin folding nucleus stabilization in the rate-limiting I reversible N transition, which occurred after the intermediate state was formed. But the amino acid residue of B-helix was very important for the folding nucleus stabilization in the transition state upon the I reversible N transition.

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.

Equilibrium unfolding of apomyoglobin by urea was investigated in the temperature range from 5 to 25 degrees C at two pH values. The thermodynamic parameters of the apomyoglobin native-unfolded state transition were determined. Conformational changes in the protein structure were monitored by tryptophan fluorescence and far UV circular dichroism. Apomyoglobin preserves its native conformation at pH 5.7 and 6.2 in the temperature range used. It was shown that the apomyoglobin stability and its unfolding cooperativity are substantially lower at 5 degrees C than at other temperatures. This fact should be taken in account at the investigation of apomyoglobin.

The influence of model negatively charged membranes on the sperm whale holomyoglobin structure at pH 6.2 has been investigated by different techniques (far and near UV circular dichroism, tryptophan fluorescence, absorbance at Soret band, differential scanning microcalorimetry and fast performance liquid chromatography). It is shown that the holomyoglobin structure undergoes a conformational transition from the native to intermediate state analogous to its apo-form. This state is characterized by the absence of a rigid tertiary structure and the native heme environment. At the same time, the content of alpha-helical secondary structure remains almost native. To change the holomyoglobin structure similarly to that of its apo-form in the presence of membranes, a higher molar phospholipids/protein ratio is required. The properties of holomyoglobin in the presence of negatively charged membranes resemble those of the molten globule state of its apo-form protein in aqueous solution. A possible functional role of the discovered non-native myoglobin state is discussed.

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.

Cytochrome b5 is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water-alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b5 as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b5 functioning is discussed.

The aqueous environment near cell membranes should be characterized by a local low pH value (due to attraction of protons by the membrane electrostatic potential) and by a local dielectric constant value (due to proximity of the water-organic boundary). It was proposed to simulate the behaviour of proteins near membranes by their behaviour in water-alcohol mixtures at moderately low pH values. Cytochrome c and retinol-binding protein are shown to be denatured at the values of pH and dielectric constant typical for the aqueous environment near the membrane surfaces. The conformational state of both these proteins under mentioned conditions is similar to the molten globule state. The basic function of the retinol-binding protein (transfer and release of vitamin A) is tightly associated with the transition of this carrier protein into the molten globule-like state, as in this case the release of retinol and protein denaturation are coupled and represent a common cooperative process.

A review of experimental data concerning physical properties of globular protein at mild denatured conditions shows that at the present time about 20th proteins were obtained in the "molten globule" state at mild denatured conditions (acid or basic pH, high temperature or moderate concentrations of guanidine hydrochloride or urea). This state is nearly as compact as the native state and has a well pronounced secondary structure but has no rigid tertiary structure. A possible role of the molten globule state in the processes of formation and of degradation of globular proteins are discussed.

The method of macroscopic diffusion with polarization optics has been applied to compare the translational diffusion coefficients for three proteins with similar three-dimensional structures in different conformational states. It has been shown that the values of diffusion coefficients obtained by the method are almost the same as those obtained by quasielastic light scattering. However this method is more available since it is less sensitive to aggregation and requires less amount of protein for investigation.

Studies are made of the pH-induced intramolecular structuring of such random copolymers as glutamic acid with leucine. It is shown that these copolymers, consisting of hydrophobic and hydrophilic residues, contain large amounts of fractions capable of acquiring non-aggregating compact structures in aqueous medium. These fractions have an approximately similar amino acid composition which does not almost depend on the composition of the initial copolymers. A decrease in the degree of ionization of side groups of glutamic acid promotes a formation of helical regions in the molecules of these fractions. At a further deionization, the helical regions collapse into compact structures stabilized by hydrophobic interactions of side groups of leucine. The compactness of the obtained structures is close to that of protein globules.