I. A. Kashparov

Apomyoglobin Stability As Dependent on Urea Concentration and Temperature at Two pH Values

Equilibrium unfolding of apomyoglobin (ApoMb) in the presence of urea was studied as dependent on the temperature (5–2°C) at two pH values (5.7 and 6.2). Thermodynamic parameters of ApoMb transition from the native to the unfolded state were estimated under various conditions. Conformational changes in ApoMb were detected by tryptophan fluorescence and far-UV circular dichroism. The ApoMb stability and the cooperativity of its unfolding at 5°C were considerably lower than at other temperatures at both pH values, where ApoMb is in the native conformation.

A new synthetic all-D-peptide with high bacterial and low mammalian cytotoxicity.

Using the synthetic alpha-helical peptide ((RLA)(2)R)(2) as a model the effect of net charge, helicity, and epimeric nature of the peptide on bactericidal potency has been examined. Both the nature and the extent of the net charge were shown to be relatively important for antibacterial activity. The loss of the structured character of the peptide resulted in reducing the activity. The all-D-peptide appeared to be a remarkably strong bacteriostatic agent with MIC <1 microM against Escherichia coli. The peptide was neither hemolytic nor cytotoxic, which in conjunction with data on its stability to enzymatic degradation makes this peptide very attractive in terms of designing new bactericidal agents on the basis of (D)((RLA)(2)R)(2).

Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (α-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, β-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (∼10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (∼600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

Apomyoglobin mutants with single point mutations at Val10 can form amyloid structures at permissive temperature

Formation of amyloid-like protein aggregates in human organs and tissues underlies many serious diseases, therefore being in the focus of numerous biochemical, medical, and molecular biological studies. So far, formation of amyloids by globular proteins has been studied mostly under conditions that strongly destabilized their native structure. Here we present our results obtained at permissive temperature by thioflavin T fluorescence, far UV CD, IR spectroscopy, and electron microscopy. We used apomyoglobin and its mutants with Ala or Phe substituted for Val10 that are structurally close to wild type apomyoglobin. It is shown that at permissive temperature the ability of the protein to form amyloids depends on the extent of its structural destabilization, but not on hydrophobicity of the substituting residue. A possible difference between amyloids formed by strongly destabilized proteins and those yielded by proteins with a slightly fluctuating native structure, as well as the stroke and infarction effect on the ability of proteins to form amyloid structures, are discussed.

Branched antimicrobial peptides

Branched peptides E(RLAR)2, E[E(RLAR)2]2, and E(KLAR)2, E[E(KLAR)2]2 were synthesized on the basis of tetrapeptides RLAR and KLAR and glutamic acid bis(pentafluorophenyl) ester. Their minimal antimicrobial concentrations were shown to decrease along with increase in branching, achieving 12 μM for Escherichia coli cells, which is comparable to antimicrobial activities of temporin, magainin, and dermaseptin. The branched peptides were found not to act on human erythrocytes.

Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

Kinetics of interactions between apomyoglobin and phospholipid membrane

The interaction of apomyoglobin and its mutant forms with phospholipid membranes was studied using tryptophan fluorescence and circular dichroism in the far UV region. It is shown that a negatively charged phospholipid membrane can have a dual effect on the structure of protein molecule upon their interaction. On the one hand, the membrane induces denaturation of the protein native structure to its intermediate state, acting as a moderate denaturing agent. On the other hand, it can stabilize the structure of unfolded protein to the same intermediate state, acting as a moderate structuring agent. The kinetics of interaction between apomyoglobin and its mutant forms and the phospholipid membrane depends on the membrane surface charge. Here the interaction rate depends on the concentration of phospholipids vesicles and stability of protein molecule, which increase with a decrease in the latter. The roles of these factors in the folding of membrane proteins and the choice of the targeted delivery pathways for protein drugs are discussed.

Antimicrobial peptides containing arginine

Tetradecapeptides (RLARLAR)2, D-(RLARLAR)2, (RLARLAA)2, and (RLGRLGR)2 were synthesized by a solid phase method using Fmoc-amino acids. The antibacterial activity of the synthesized peptides was studied against Escherichia coli cells. The minimum inhibitory concentration (MIC) was, correspondingly, 3, 1, 3, and 12 μM, which is comparable with MIC of such natural antimicrobial peptides as temporin, magainin, and dermaseptin. It was found that all of the synthesized peptides have no effect on human erythrocytes and rat thymocytes. The peptides form α-helices in 30% trifluoroethanol and in 2.5 mM SDS, which have amphipathic structure.

Dataset concerning GroEL chaperonin interaction with proteins

GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020[1]), and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin) and denatured (lysozyme, α-lactalbumin and pepsin) proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

The Effect of Protein Stability on Interactions of Apomyoglobin Forms with Phospholipid Membranes

The protein structure can be strongly influenced by phospholipid membranes. As it follows from our papers, apomyoglobin structure undergoes a transition from the native to some intermediate state upon interaction with small negatively charged phospholipid vesicles acting as a moderately denaturing agent. In this work, interaction of apomyoglobin and its mutant forms with artificial membranes is studied by tryptophan fluorescence and CD in the far UV-region. It is shown that a negatively charged phospholipid membrane can structure the unfolded protein into the same intermediate state. The nature of this phenomenon consists in selective stabilization of the intermediate state structure. The rate of interactions between apomyoglobin mutant forms and phospholipid membranes depends mainly on the protein molecule stability, as well as on the charge of the membrane surface and the phospholipid vesicles concentration. This rate increases with decreasing protein stability. 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. This work was supported partly by the Howard Hughes Medical Institute Award 55005607 to A.V. Finkelstein, by the RAS Program “Molecular and Cellular Biology”, by Federal Agency for Science and Innovations 02.740.11.0295, and Program of Scientific Schools 2791.2008.4.