Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A.

Single-stranded-DNA-binding proteins (SSBs) are required for numerous genetic processes ranging from DNA synthesis to the repair of DNA damage, each of which requires binding with high affinity to ssDNA of variable base composition. To gain insight into the mechanism of sequence-nonspecific binding of ssDNA, NMR chemical shift and (15)N relaxation experiments were performed on an isolated ssDNA-binding domain (RPA70A) from the human SSB replication protein A. The backbone (13)C, (15)N, and (1)H resonances of RPA70A were assigned for the free protein and the d-CTTCA complex. The binding-induced changes in backbone chemical shifts were used to map out the ssDNA-binding site. Comparison to results obtained for the complex with d-C(5) showed that the basic mode of binding is independent of the ssDNA sequence, but that there are differences in the binding surfaces. Amide nitrogen relaxation rates (R(1) and R(2)) and (1)H-(15)N NOE values were measured for RPA70A in the absence and presence of d-CTTCA. Analysis of the data using the Model-Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA-binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, we propose that the mechanism of sequence-nonspecific binding of ssDNA involves dynamic remodeling of the binding surface.

Crumpled structure of the custom hydrophobic lytic peptide cecropin B3.

The solution structure of a custom lytic peptide, cecropin B3 (CB3), having two identical hydrophobic segments on both the N- and C-termini, was investigated by two-dimensional NMR spectroscopy. The need to determine the structure of this peptide is rooted in its specific ability to lyse lipid layers that have a high content of anionic lipid. The lytic activities of CB3 on cell membranes including cancer cells and bacteria is found to be less than cecropin B1. The results show that CB3 has four discrete segments forming alpha helical structures. The crumpled structure of CB3 provides evidence for the lysis of the lipid layer being via a pathway that differs from pore formation. The results in this study provide strong clues towards a rational design for a potent antimicrobial and antitumor peptide.

Conformational study of a custom antibacterial peptide cecropin B1: implications of the lytic activity.

Cecropin B1 (CB1) with two amphipathic alpha-helical segments is a derivative of the natural antibacterial peptide, cecropin B. The assays of cell lysis show that, compared with cecropin A (CA), CB1 has a similar ability to lyse bacteria with a higher potency (two- to six-fold higher) in killing cancer cells. The difference may be due to the fact that the peptides possess different structures and sequences. In this study, the solution structure of CB1 in 20% hexafluoroisopropanol was determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The (1)H NMR resonances were assigned. A total of 350 inter-proton distances were used to calculate the solution structure of CB1. The final ensemble structures were well converged, showing the minimum root mean square deviation. The results indicate that CB1 has two stretches of helices spanning from residues 3 to 22 and from residues 26 to 33, which are connected by a hinge section formed by Gly-23 and Pro-24. Lys-25 is partially incorporated in the hinge region. The bent angle between two helical segments located in two planes was between 100 and 110 degrees. With comparisons of the known NMR structure of CA and its activities on bacteria and cancer cells, the structure-function relationship of the peptides is discussed.

Specificity of helix-induction by 2,2,2-trifluoroethanol in polypeptides.

The specificity of helix-induction in polypeptides by 2,2,2-trifluoro ethanol (TFE) is studied using an all beta-sheet protein such as cardiotoxin analogue I (CTX I) from the Taiwan Cobra (Naja naja atra) and a homopolymer such as poly-L-lysine. It is found that alcohols including TFE can 'non-specifically' induce helix at high concentrations both in CTX I and polylysine at neutral pH. However, among the alcohols used, only TFE could transform the heat-induced beta-sheet conformation of polylysine at pH 11.5 into an alpha-helix. The beta-sheet to alpha-helix conversion in polylysine (in the beta-sheet conformation) occurs even at very low concentrations of TFE (< 5% v/v). In addition, experiments on the effect(s) of TFE on the denatured and reduced CTX I (rCTX I) indicate the helix-induction in rCTX I takes place at low TFE concentrations (< 20% v/v). The results of this study hint at the possible influence of disulfide bridges on the induction of helix by TFE.

Non-specific helix-induction in charged homopolypeptides by alcohols.

The specificity/non-specificity of helix-induction in charged homopolymers such as polylysine and polyglutamic acid, at neutral pH, by various alcohols namely 2,2,2-trifluoroethanol (TFE), methanol, ethanol and 1-propanol is studied. It is found that all the alcohols used, non-specifically induced helical conformation at high concentrations. In addition, the effect(s) of TFE on an all beta-sheet protein, such cardiotoxin analogue I (CTX I) from the Taiwan Cobra (Naja naja atra) is also studied. Evaluation of the helix propensity in the amino-acid sequence of CTX I using helix-coil algorithm, AGADIR, shows a total of 1.15% helical content in the protein. In CTX I, helical conformation is found to be induced at high concentrations of TFE (> or = 70% v/v). Interestingly, upon denaturation and reduction of disulfide bridges in CTX I, helix is found to be induced even at low concentrations of TFE (> or = 20% v/v). The results of this study hints at the possible influence of native tertiary structural interactions and disulfide bridges in the induction of helix by TFE.

Conformation of a protein kinase C substrate NG(28-43), and its analog in aqueous and sodium dodecyl sulfate micelle solutions.

A peptide corresponding to the neuronal protein neurogranin (NG) residues 28-43, NG(28-43), and its analog, [A35]NG(28-43), have been investigated by NMR, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies. The peptides existed in aqueous solution predominantly in random form. However, a nascent helical structure was detected in the central region of the parent peptide from NMR data. Furthermore, a helical structure can be detected for both peptides with greater induced secondary structure for the parent peptide in the presence of sodium dodecyl sulfate (SDS) micelle. The formation of micelles for SDS was confirmed by results from EPR as well as 13C NMR. As shown by CD experiments, helical conformer was induced for NG(28-43) in vesicular solution containing phosphatidyl serine (PS), whereas no helix can be discerned for the peptide in phosphatidyl choline (PC)-containing vesicular solution. Together with the induction of the peptide into helix in SDS micellar solution as suggested by both NMR and CD data, these results underscored the electrostatic contribution to the interaction of the PKC substrate peptides and proteins with membrane. According to NMR and CD data, a dynamic equilibrium existed between free and micelle-bound states for the peptide. Moreover, proton-deuterium exchange results and SDS-induced linewidth broadening of proton resonances allowed delineation of the orientation of the amphipathic helix on the surface of SDS micelle. The result was supported by spin label experiments that indicated F35 of NG(28-43) interacted strongly with the hydrocarbon interior of micelle. Based on the experimental findings, a working model was proposed that attempted to partly explain the roles played by the nonpolar amino acid near the phosphorylation site, by the negatively charged phospholipids, and by the basic amino acids of the substrate.

Acetonitrile-induced conformational transitions in poly-L-lysine.

The effect of acetonitrile on the random coil, alpha-helix and beta-sheet conformations induced in poly-L-lysine is studied. It is found that acetonitrile at higher concentrations transforms the backbone of polylysine from a random coil to a helical conformation. Addition of acetonitrile to polylysine (pH 11.5) in the alpha-helix conformation, induces conformational changes in two stages. At concentrations below 60% v/v, acetonitrile stabilizes the helical conformation and at higher concentrations (> 70% v/v), it destabilizes the helix. beta-sheet-->alpha-helix-->random coil conformational transitions are found to occur when polylysine in the heat-induced conformation is titrated with acetonitrile. The possible mechanism(s) of action of acetonitrile in inducing these structural transitions is discussed.

Snake venom cardiotoxins-structure, dynamics, function and folding.

Snake cardiotoxins are highly basic (pI > 10) small molecular weight (approximately 6.5 kDa), all beta-sheet proteins. They exhibit a broad spectrum of interesting biological activities. The secondary structural elements in these toxins include antiparallel double and triple stranded beta-sheets. The three dimensional structures of these toxins reveal an unique asymmetric distribution of the hydrophobic and hydrophilic amino acids. The 3D structures of closely related snake venom toxins such as neurotoxins and cardiotoxin-like basic proteins (CLBP) fail to show similar pattern(s) in the distribution of polar and nonpolar residues. Recently, many novel biological activities have been reported for cardiotoxins. However, to-date, there is no clear structure-function correlation(s) available for snake venom cardiotoxins. The aim of this comprehensive review is to summarize and critically evaluate the progress in research on the structure, dynamics, function and folding aspects of snake venom cardiotoxins.

Induction of helical conformation in all beta-sheet proteins by trifluoroethanol.

The effect of 2,2,2-Trifluoroethanol (TFE) on the structure of five all beta-sheet proteins, isolated from the venom of the Taiwan cobra (Naja naja atra), is studied. In all the toxins used, it is observed that significant amount of alpha-helix is induced at higher concentrations of TFE. In all these proteins, the induction of helical conformation and disruption of the tertiary structure seem to occur simultaneously. The structural transitions induced by TFE in reduced and denatured protein appear to be different from those observed in the native protein(s). In our opinion, the findings reported herein could have significant implications on research in the area of protein folding.

2,2,2-Trifluoroethanol induces helical conformation in an all beta-sheet protein.

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all beta-sheet protein, cardiotoxin analogue II (CTX II), from the Taiwan cobra (Naja naja atra) is studied. Using circular dichroism studies, it is found that higher concentrations of TFE induced a structural transition from beta-sheet to alpha-helix, both in the native state (nCTX II) and in denatured but not disulfide reduced CTX II (dCTX II) samples. The beta-sheet to alpha-helix conversion is shown to be cooperative. However, in denatured and reduced CTX II (rCTX II), a TFE transforms a portion(s) of the protein backbone a random coil to an alpha-helical conformation. Based on the solution structure of CTX II and the physical property of TFE, a possible mechanism for the observed backbone structural transitions induced by TFE is discussed. The results described in this paper question the significance of the structure of the "molten globule" intermediate(s) obtained in organic solvents such as TFE.

NMR and dynamical simulated annealing studies on the solution conformation of urotensin II.

We determined the structure in solution of the vaso-constrictor hormone urotensin II (dodecapeptide) using nuclear magnetic resonance spectroscopy. Complete assignment of all proton resonances has been achieved and the structural information has been obtained from the interproton distance measurements derived from the nuclear Overhauser enhancement data. A combination of distance geometry and dynamical simulated annealing techniques was used to calculate the structure in solution. Nine resultant structures with fewer distance constraint violations were selected that satisfy the experimental restraints very well. The conformation of the molecule in the cyclic hexapeptide segment (core region) is well-defined whereas the N-terminal segment is disordered. This result correlates very well with the earlier predictions about the biologically active and inactive roles played by the core and the N-terminal segment respectively.