Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.     

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by (13)C-NMR.

We have examined the (13)C-NMR spectra of [3-(13)C] Ala-labeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal alpha helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal alpha helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mM NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) (13)C signals from the C-terminal alpha helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal alpha helix is deleted or site-directed mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.     

Role of the C-terminal helix 9 in the stability and ligandin function of class alpha glutathione transferase A1-1

Helix 9 at the C-terminus of class alpha glutathione transferase (GST) polypeptides is a unique structural feature in the GST superfamily. It plays an important structural role in the catalytic cycle. Its contribution toward protein stability/folding as well as the binding of nonsubstrate ligands was investigated by protein engineering, conformational stability, enzyme activity, and ligand-binding methods. The helix9 sequence displays an unfavorable propensity toward helix formation, but tertiary interactions between the amphipathic helix and the GST seem to contribute sufficient stability to populate the helix on the surface of the protein. The helix's stability is enhanced further by the binding of ligands at the active site. The order of ligand-induced stabilization increases from H-site occupation, to G-site occupation, to the simultaneous occupation of H- and G-sites. Ligand-induced stabilization of helix9 reduces solvent accessible hydrophobic surface by facilitating firmer packing at the hydrophobic interface between helix and GST. This stabilized form exhibits enhanced affinity for the binding of nonsubstrate ligands to ligandin sites (i.e., noncatalytic binding sites). Although helix9 contributes very little toward the global stability of hGSTA1-1, its conformational dynamics have significant implications for the protein's equilibrium unfolding/refolding pathway and unfolding kinetics. Considering the high concentration of reduced glutathione in human cells (about 10 mM), the physiological form of hGSTA1-1 is most likely the thiol-complexed protein with a stabilized helix9. The C-terminus region (including helix9) of the class alpha polypeptide appears not to have been optimized for stability but rather for catalytic and ligandin function.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Distinct structural changes detected by X-ray fiber diffraction in stabilization of F-actin by lowering pH and increasing ionic strength

Lowering pH or raising salt concentration stabilizes the F-actin structure by increasing the free energy change associated with its polymerization. To understand the F-actin stabilization mechanism, we studied the effect of pH, salt concentration, and cation species on the F-actin structure. X-ray fiber diffraction patterns recorded from highly ordered F-actin sols at high density enabled us to detect minute changes of diffraction intensities and to precisely determine the helical parameters. F-actin in a solution containing 30 mM NaCl at pH 8 was taken as the control. F-actin at pH 8, 30 to 90 mM NaCl or 30 mM KCl showed a helical symmetry of 2.161 subunits per turn of the 1-start helix (12.968 subunits/6 turns). Lowering pH from 8 to 6 or replacing NaCl by LiCl altered the helical symmetry to 2.159 subunits per turn (12.952/6). The diffraction intensity associated with the 27-A meridional layer-line increased as the pH decreased but decreased as the NaCl concentration increased. None of the solvent conditions tested gave rise to significant changes in the pitch of the left-handed 1-start helix (approximately 59.8 A). The present results indicate that the two factors that stabilize F-actin, relatively low pH and high salt concentration, have distinct effects on the F-actin structure. Possible mechanisms will be discussed to understand how F-actin is stabilized under these conditions.     

Cooperative helix stabilization by complex Arg-Glu salt bridges

Among the interactions that stabilize the native state of proteins, the role of electrostatic interactions has been difficult to quantify precisely. Surface salt bridges or ion pairs between acidic and basic side chains have only a modest stabilizing effect on the stability of helical peptides or proteins: estimates are roughly 0.5 kcal/mol or less. On the other hand, theoretical arguments and the occurrence of salt bridge networks in thermophilic proteins suggest that multiple salt bridges may exert a stronger stabilizing effect. We show here that triads of charged side chains, Arg(+)-Glu(-)-Arg(+) spaced at i,i+4 or i,i+3 intervals in a helical peptide stabilize alpha helix by more than the additive contribution of two single salt bridges. The free energy of the triad is more than 1 kcal/mol in excess of the sum of the individual pairs, measured in low salt concentration (10 mM). The effect of spacing the three groups is severe; placing the charges at i,i+4 or i,i+3 sites has a strong effect on stability relative to single bridges; other combinations are weaker. A conservative calculation suggests that interactions of this kind between salt bridges can account for much of the stabilization of certain thermophilic proteins.     

Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Melting of cross-linked DNA v. cross-linking effect caused by local stabilization of the double helix

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this "cross-linking effect" (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the "cross-linking effect" (18 - 20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20- 40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Conformational change of poly-N epsilon-glutaryl-L-lysine and poly-N epsilon-succinyl-L-lysine in aqueous salt solutions

The conformational changes of poly-N^ε-glutaryl-L -lysine (PGL) and poly-N^ε-succinyl-L -lysine (PSL) in various salt solutions were studied by use of ORD and potentiometric titration measurements. The addition of alkali metal salts to the fully ionized PGL or PSL solution caused helix formation. The helical content of the polymers increases in the following sequences: at salt concentration 0–2 M, CsCl < KCl < LiCl < NaCl; and at 2–3 M, LiCl < CsCl < KCl ～ NaCl. The preferential binding of the solvent components with various alkali metal salts of PGL or PSL was measured in LiCl, NaCl, and KCl solutions by means of equilibrium dialysis and differential refractometry. It was found that with increasing salt concentration, the polymers were preferentially hydrated in NaCl and KCl soultions; however the salt was preferentially bound to the polymers in LiCl solution. Such preferential binding was suggested to be closely related to conformational change. The addition of CaCl₂ to polymer solutions led to the stabilization of the helical structure of PGL or PSL.     

Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation

The L7/12 stalk of the large subunit of bacterial ribosomes encompasses protein L10 and multiple copies of L7/12. We present crystal structures of Thermotoga maritima L10 in complex with three L7/12 N-terminal-domain dimers, refine the structure of an archaeal L10E N-terminal domain on the 50S subunit, and identify these elements in cryo-electron-microscopic reconstructions of Escherichia coli ribosomes. The mobile C-terminal helix alpha8 of L10 carries three L7/12 dimers in T. maritima and two in E. coli, in concordance with the different length of helix alpha8 of L10 in these organisms. The stalk is organized into three elements (stalk base, L10 helix alpha8-L7/12 N-terminal-domain complex, and L7/12 C-terminal domains) linked by flexible connections. Highly mobile L7/12 C-terminal domains promote recruitment of translation factors to the ribosome and stimulate GTP hydrolysis by the ribosome bound factors through stabilization of their active GTPase conformation.     

Equilibrium oxygen binding to human hemoglobin cross-linked between the alpha chains by bis(3,5-dibromosalicyl) fumarate

Oxygen equilibrium curves of human hemoglobin Ao (HbAo) and human hemoglobin cross-linked between the alpha chains (alpha alpha Hb) by bis(3,5-dibromosalicyl) fumarate were measured as a function of pH and chloride or organic phosphate concentration. Compared to HbAo, the oxygen affinity of alpha alpha Hb was lower, cooperativity was maintained, although slightly reduced, and all heterotropic effects were diminished. The major effect of alpha alpha-cross-linking appears to be a reduction of the oxygen affinity of R-state hemoglobin under all conditions. However, while the oxygen affinity of T-state alpha alpha Hb was slightly reduced at physiologic chloride concentration and in the absence of organic phosphates, KT was the same for both hemoglobins in the presence of 2,3-diphosphoglycerate (or high salt) and higher for alpha alpha Hb in the presence of inositol hexaphosphate. The reduced O2 affinity arises from smaller binding constants for both T- and R-state alpha alpha Hb rather than through stabilization of the low affinity conformation. All four Adair constants could be determined for alpha alpha Hb under most conditions, but a3 could not be resolved for HbAo without constraining a4, suggesting that the cross-link stabilizes triply ligated intermediates of hemoglobin.     

Cosolvent,ions, and temperature effects on the structural properties of cecropin A‐Magainin 2 hybrid peptide in solutions

Antimicrobial peptides are promising alternative to traditional antibiotics and antitumor drugs for the battle against new antibiotic resistant bacteria strains and cancer maladies. The study of their structural and dynamics properties at physiological conditions can help to understand their stability, delivery mechanisms, and activity in the human body. In this article, we have used molecular dynamics simulations to study the effects of solvent environment, temperature, ions concentration, and peptide concentration on the structural properties of the antimicrobial hybrid peptide Cecropin A–Magainin 2. In TFE/water mixtures, the structure of the peptide retained α‐helix contents and an average hinge angle in close agreement with the experimental NMR and CD measurements reported in literature. Compared to the TFE/water mixture, the peptide simulated at the same ionic concentration lost most of its α‐helix structure. The increase of peptide concentration at both 300 and 310 K resulted in the peptide aggregation. The peptides in the complex retained the initial N‐ter α‐helix segment during all the simulation. The α‐helix stabilization is further enhanced in the high salt concentration simulations. The peptide aggregation was not observed in TFE/water mixture simulations and, the peptide aggregate, obtained from the water simulation, simulated in the same conditions did dissolve within few tens of nanoseconds. The results of this study provide insights at molecular level on the structural and dynamics properties of the CA‐MA peptide at physiological and membrane mimic conditions that can help to better understand its delivery and interaction with biological interfaces. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 1–14, 2015.     

The effects of the side chains of hydrophobic aliphatic amino acid residues in an amphipathic polypeptide on the formation of alpha helix and its association

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.     

Recombinant collagen studies link the severe conformational changes induced by osteogenesis imperfecta mutations to the disruption of a set of interchain salt bridges

The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the alpha1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly --> Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen.     

Role of stabilization centers in 4 helix bundle proteins

Stabilization centers (SCs) were shown to play an important role in preventing decay of three-dimensional protein structures. These residue clusters, stabilized by cooperative long-range interactions, were proposed to serve as anchoring points for arranging secondary structure elements. In all-alpha proteins, SC elements appear less frequently than in all-beta, alpha/beta, and alpha+beta proteins suggesting that tertiary structure formation of all-alpha proteins is governed by different principles than in other protein classes. Here we analyzed the relation between the formation of stabilization centers and the inter-axial angles (Omega) of alpha-helices in 4 helix bundle proteins. In the distance range, where dipoles have dominant effect on the helix pair arrangement, those helix pairs, where residues from both helices participate in SC elements, appear as parallel more frequently than those helices where no SC elements are present. For SC containing helix pairs, the energetic difference between the parallel and anti-parallel states decreases considerably from 1.1 kcal/mol to 0.4 kcal/mol. Although the observed effect is weak for more distant helices, a competition between the SC element formation and the optimal dipole-dipole interaction of alpha-helices is proposed as a mechanism for tertiary structure formation in 4 helix bundle proteins. The SC-forming potential of different arrangements as well as the pitfalls of the SC definition are also discussed.     

Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form

The crystal structure of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-GK) in a dimeric form was determined at a resolution of 2.4 A. This is the first crystal structure of a hyperthermophilic glycerol kinase. The overall structure of the Tk-GK dimer is very similar to that of the Escherichia coli glycerol kinase (Ec-GK) dimer. However, two dimers of Ec-GK can associate into a tetramer with a twofold axis, whereas those of Tk-GK cannot. This may be the reason why Tk-GK is not inhibited by fructose 1,6-bisphosphate, because the fructose 1,6-bisphosphate binding site is produced only when a tetrameric structure is formed. Differential scanning calorimetry analyses indicate that Tk-GK is a highly thermostable protein with a melting temperature (T(m)) of 105.4 degrees C for the major transition. This value is higher than that of Ec-GK by 34.1 degrees C. Comparison of the crystal structures of Tk-GK and Ec-GK indicate that there is a marked difference in the number of ion pairs in the alpha16 helix. Four ion pairs, termed IP1-IP4, are formed in this helix in the Tk-GK structure. To examine whether these ion pairs contribute to the stabilization of Tk-GK, four Tk-GK and four Ec-GK derivatives with reciprocal mutations at the IP1-IP4 sites were constructed. The determination of their stabilities indicates that the removal of each ion pair does not affect the stability of Tk-GK significantly, whereas the mutations designed to introduce one of these ion pairs stabilize or destabilize Ec-GK considerably. These results suggest that the ion pairs in the alpha16 helix contribute to the stabilization of Tk-GK in a cooperative manner.     

Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes

Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.     

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na⁺. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.