Natalia G. Esipova

Mechanism of Lac repressor switch-off: orientation of the Lac repressor DNA-binding domain is reversed upon inducer binding

Lac repressors DNA‐binding domains contain helix‐turn‐helix motif which, though similar to those of phage λ Cro protein, are oriented differently with respect to DNA: in the specific complexes with Lac operator, N termini of the repressors subunits are facing inwards. We demonstrate that, in the presence of an inducer, the repressors N termini cross‐link to the operators outermost nucleotides. We suggest that the inducer fixes the repressors DNA‐binding domains in the Cro‐type configuration and thus garbles its recognition surface. Since the Cro‐type configuration is perfectly suitable for binding the DNA, this also explains how the switched‐off repressor retains its non‐specific DNA‐binding.

Comparative study of thermostability and structure of close homologues--barnase and binase.

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2-6 have been determined. The barnase heat denaturation (pH 2.8-5.5) proceeds according to the all-or-none principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.

Natural polypeptides in left‐handed helical conformation A circular dichroism study of the linker histones’ C‐terminal fragments and β‐endorphin

Circular dichroism has been used to investigate the histone HI and H5 C‐terminal fragments and β‐endorphin conformation. It has been shown that in aqueous solution these polypeptides preferably adopt the left‐handed helical conformation of the poly‐l‐proline II type. A break in the linear temperature dependence of the CD value was found in the temperature interval between 50 and 55°C. It was proposed to be due to non‐cooperative disordering of the conformation caused by the destruction of the hydration shell.

Comprehensive statistical analysis of residues interaction specificity at protein–protein interfaces

We calculated interchain contacts on the atomic level for nonredundant set of 4602 protein‐protein interfaces using an unbiased Voronoi‐Delaune tessellation method, and made 20×20 residue contact matrixes both for homodimers and heterocomplexes. The area of contacts and the distance distribution for these contacts were calculated on both the residue and the atomic levels. We analyzed residue area distribution and showed the existence of two types of interresidue contacts: stochastic and specific. We also derived formulas describing the distribution of contact area for stochastic and specific interactions in parametric form. Maximum pairing preference index was found for Cys‐Cys contacts and for oppositely charged interactions. A significant difference in residue contacts was observed between homodimers and heterocomplexes. Interfaces in homodimers were enriched with contacts between residues of the same type due to the effects of structure symmetry. Proteins 2007.

Hierarchy of the Interaction Energy Distribution in the Spatial Structure of Globular Proteins and the Problem of Domain Definition

An algorithm for determining of protein domain structure is proposed. Domain structures resulted from the algorithm application have been obtained and compared with available data. The method is based on entirely physical model of van der Waals interactions that reflects as illustrated in this work the distribution of electron density. Various levels of hierarchy in the protein spatial structure are discerned by analysis of the energy interaction between structural units of different scales. Thus the level of energy hierarchy plays role of sole parameter, and the method obviates the use of complicated geometrical criteria with numerous fitting parameters. The algorithm readily and accurately locates domains formed by continuous segments of the protein chain as well as those comprising non-sequential segments, sets no limit to the number of segments in a domain. We have analyzed 309 protein structures. Among 277 structures for which our results could be compared with the domain definitions made in other works, 243 showed complete or partial coincidence, and only in 34 cases the domain structures proved substantially different. The domains delineated with our approach may coincide with reference definition at different levels of the globule hierarchy. Along with defining the domain structure, our approach allows one to consider the protein spatial structure in terms of the spatial distribution of the interaction energy in order to establish the correspondence between the hierarchy of energy distribution and the hierarchy of structural elements.

Representation of amino acid sequences in terms of interaction energy in protein globules

We suggest a new simple approach for comparing the primary structure of proteins and their spatial structure. It relies on the one‐to‐one correspondence between each residue of the polypeptide chain and the energy of van der Waals interactions between the regions of the native globule flanking this residue. The method obviates the sophisticated geometrical criteria for estimating similarity between spatial structures. Besides, it permits one to analyze structural units of different scale.

Analysis of forces that determine helix formation in α‐proteins

A model for prediction of α‐helical regions in amino acid sequences has been tested on the mainly‐α protein structure class. The modeling represents the construction of a continuous hypothetical α‐helical conformation for the whole protein chain, and was performed using molecular mechanics tools. The positive prediction of α‐helical and non‐α‐helical pentapeptide fragments of the proteins is 79%. The model considers only local interactions in the polypeptide chain without the influence of the tertiary structure. It was shown that the local interaction defines the α‐helical conformation for 85% of the native α‐helical regions. The relative energy contributions to the energy of the model were analyzed with the finding that the van der Waals component determines the formation of α‐helices. Hydrogen bonds remain at constant energy independently whether α‐helix or non‐α‐helix occurs in the native protein, and do not determine the location of helical regions. In contrast to existing methods, this approach additionally permits the prediction of conformations of side chains. The model suggests the correct values for ∼60% of all χ‐angles of α‐helical residues.

A tetrapeptide-based method for polyproline II-type secondary structure prediction

We describe a new method for polyproline II‐type (PPII) secondary structure prediction based on tetrapeptide conformation properties using data obtained from all globular proteins in the Protein Data Bank (PDB). This is the first method for PPII prediction with a relatively high level of accuracy (∼60%). Our method uses only frequencies of different conformations among oligopeptides without any additional parameters. We also attempted to predict α‐helices and β‐strands using the same approach. We find that the application of our method reveals interrelation between sequence and structure even for very short oligopeptides (tetrapeptides). Proteins 2005.

Scanning microcalorimetry and circular dichroism study of melting of the natural polypeptides in the left-handed helical conformation

It has been shown that in aqueous solution histone H1 and H5 C-terminal fragments and peptide hormones β-endorphin and ACTH adopt preferably the left-handed helical conformation of the poly-l-proline II type. Scanning microcalorimetry and circular dichroism have been used to show that the linear temperature dependence of CD maximum amplitude and partial heat capacity value are broken in the temperature interval between 50 and 60°C, after which [C]p reaches the constant level. It was proposed to be due to noncooperative disordering of the conformation caused by the destruction of the polypeptide hydration shell.

HEAT DENATURATION OF PEPSINOGEN IN A WATER-ETHANOL MIXTURE

The effect of ethanol and pH on thermodynamic parameters and cooperativity of pepsinogen heat denaturation was studied by scanning microcalorimetry. Addition of 20% ethanol decreases the protein denaturation temperature by 10.7°C at pH 6.4 and 15.8°C at pH 8.0. It also decreases the denaturation heat capacity increment from 5.8 to 4.2 kcal/K·mol. The dependences of calorimetric denaturation enthalpy on denaturation temperature both in water and 20% ethanol are linear and intersect at about 95°C. In 20% ethanol the pH shift from 5.9 to 8.0 results in a decreased number of cooperative domains in pepsinogen. This process causes no changes either in the secondary structure or in the local surroundings of aromatic amino acids. It is concluded that ethanol addition does not affect the cooperativity of pepsinogen denaturation substantially until the pH change provokes redistribution of charges in the protein molecule.