A strained DNA binding helix is conserved for site recognition,folding nucleation,and conformational modulation

Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 3₁₀ helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10⁴‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432–443, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Molecular dynamics (MD) simulations of VIP and PACAP27

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide‐27 (PACAP27) are members of the secretin‐glucagon family containing 28 and 27 residues, respectively. NMR spectroscopy studies suggest that the N‐terminus exhibit consecutive β‐turns whereas the central and C‐terminal parts of the VIP molecule have been characterized as being two α‐helices. In contrast, similar studies carried out on PACAP suggest that the shortest active peptide segment PACAP27 in the presence of trifluoroethanol (TFE) exhibits a disordered N‐terminal domain followed by a α‐helix expanding residues 9–26 with a discontinuity between residues 20 and 21. In the present study, a series of MD trajectories of VIP and PACAP27 were carried out using two different implicit models of the solvent: the Generalized Born that use an effective Born radii described by Onufriev, Bashford, and Case (GB^OBC) and the Hawkins, Cramer, and Truhlar approximation (GB^HCT) and two different force fields: AMBER ff99 and a modified version of the latter described by Sorin and Pande (Biophys J 2005, 88, 2472‐2493), ff99SP. Comparison of the structures obtained from the MD trajectories and those derived from the NMR studies in the literature indicates that the GB^OBC method is more efficient in the exploration of the conformational space and presents a higher agreement with the experimental structure of VIP and PACAP27 in TFE. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 391–400, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Importance of the spatial display of charged residues in heparin–peptide interactions

Many studies have examined consensus sequences required for protein‐glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the α‐helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein‐heparin binding interactions when the charged residues are aligned linearly. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 290–298, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transfer

A fluorescently labeled 20‐residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E₂₀) was used to study structural changes which occur in E₂₀ as it co‐aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o‐aminobenzamide donor at the N‐terminus and 3‐nitrotyrosine acceptor at the C‐terminus of E₂₀. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from α‐helical monomers to aggregated β‐sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time‐resolved and steady‐state RET measurements reveal four dominant E₂₀ conformations: (1) a partially collapsed conformation (24 Å donor–acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 Å donor–acceptor distance), (3) a minor partially collapsed conformation (22 Å donor‐acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 Å donor–acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom‐level structural details of soluble oligomer and aggregated states of proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 299–317, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Helix‐coil energetics for helix formers and breakers reflect context and temperature: Mutants of helically robust,guest‐sensitive homopeptide hosts

The natural amino acids are primarily helix breakers at the low assignment temperatures characteristic of many studies, but recent genomic analyses of thermophilic proteins suggest that at high temperatures, some breakers may become strong helix formers. Moreover, the breaker/former inventory has not been previously characterized at the physiologically relevant temperature of 37°C. The versatility of ¹³C?O NMR chemical shifts as helicity reporters allows construction of two mutant peptide series, tailored to expand the range of temperature assignments for helical propensities and derived from the core hosts ^tL‐Ala₉XxxAla₉‐^tL and ^tL‐AlaNva₄XxxNva₄Ala₉‐^tL, Nva = norvaline. For three limiting guests Xxx, the helix former Nva and the breakers Gly and Pro, we report w_Xxx[T] assignments at seven temperatures from 2 to 80°C, validating our reasoning and paving the way for assignment of a definitive w_Xxx[T] data‐base. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 311–320, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti‐inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin^50–59 sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well‐defined conformation in methanol, its structure was distinct from the corresponding 50–59 fragment in the native ubiquitin molecule. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 423–431, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Thermal stability and conformational structure of salmon calcitonin in the solid and liquid states

Salmon calcitonin (sCT) was selected as a model protein drug for investigating its intrinsic thermal stability and conformational structure in the solid and liquid states by using a Fourier transform infrared (FT‐IR) microspectroscopy with or without utilizing thermal analyzer. The spectral correlation coefficient (r) analysis between two second‐derivative IR spectra was applied to quantitatively estimate the structural similarity of sCT in the solid state before and after different treatments. The thermal FT‐IR microspectroscopic data clearly evidenced that sCT in the solid state was not effected by temperature and had a thermal reversible property during heating–cooling process. Moreover, the high r value of 0.973 or 0.988 also evidenced the structural similarity of solid‐state sCT samples before and after treatments. However, sCT in H₂O exhibited protein instability and thermal irreversibility after incubation at 40°C. The temperature‐induced conformational changes of sCT in H₂O was occurred to transform the α‐helix/random coil structures to β‐sheet structure and also resulted in the formation of intramolecular and intermolecular β‐sheet structures. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 200–207, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Terminal sidechain packing of a designed β‐hairpin influences conformation and stability

While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔG_U = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Interaction of α‐gliadin with polyanions: Design considerations for sequestrants used in supportive treatment of celiac disease

Copolymers of sodium 4‐styrene sulfonate (SS) and hydroxyethyl methacrylate (HEMA) were investigated as sequestrants of α‐gliadin, a gluten protein, for the treatment of gluten intolerance. The interactions of α‐gliadin with poly(SS) and poly(HEMA‐co‐SS) with 9 and 26 mol% SS content were studied at gastric (1.2) and intestinal (6.8) pH using circular dichroism and measurements of turbidity, dynamic light scattering and zeta potential. The interactions and their influence on α‐gliadin secondary and aggregated structures depended mainly on the ratio of polymer negative and protein positive charges at pH 1.2, and on polymer SS content at polymer concentrations providing in excess of negative charges at either pH. Poly(SS) could not form complex particles with α‐gliadin in a sufficient excess of negative charges. Copolymerization with HEMA enhanced the formation of complex particles. Poly(HEMA‐co‐SS) with intermediate SS content was found to be the most effective sequestrant for α‐gliadin. This study provides insight into design considerations for polymer sequestrants used in the supportive treatment of celiac disease. © 2009 Wiley Periodicals, Inc. Biopolymers 93:418–428, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The effect of the side chain length of Asp and Glu on coordination structure of Cu2+ in a de novo designed protein

Metal ions in proteins are important not only for the formation of the proper structures but also for various biological activities. For biological functions such as hydrolysis and oxidation, metal ions often adopt unusual coordination structures. We constructed a stable scaffold for metal binding to create distorted metal coordination structures. A stable four stranded α‐helical coiled‐coil structure was used as the scaffold, and the metal binding site was in the cavity created at the center of the structure. Two His residues and one Asp or Glu residue were used to coordinate the metal ions, AM2D and AM2E, respectively. Cu²⁺ bound to AM2D with an equatorial planar coordination structure with two His, one Asp, and H₂O as detected by electron spin resonance and UV spectral analyzes. On the other hand, Cu²⁺ had a slightly distorted square planar structure when it bound two His and Glu in AM2E, due to the longer side‐chain of the Glu residue as compared to the Asp residue. Computational analysis also supported the distorted coordination structure of Cu²⁺ in AM2E. This construct should be useful to create various coordinations of metal ions for catalytic functions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 907–916, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

3(10)-Helix adjoining alpha-helix and beta-strand: sequence and structural features and their conservation

Does the amino acid use at the terminal positions of an α‐helix become altered depending on the context—more specifically, when there is an adjoining 3₁₀‐helix, and can a single helical cylinder encompass the resultant composite helix? An analysis of 138 and 107 cases of 3₁₀–α and α–3₁₀ composite helices, respectively, found in known protein structures indicate that the secondary structural element occurring first imposes its characteristics on the sequence of the structural element coming next. Thus, when preceded by a 3₁₀‐helix, the preference of proline to occur at the N1 position of an α‐helix is shifted to the N2 position, a typical characteristic of the C‐terminal capping of the 3₁₀‐helix. When an α‐ or a 3₁₀‐helix leads into a helix of the other type, there is a bend at the junction, especially for the 3₁₀–α composite, with the two junction residues facing inward and buried within the structure. Thus a single helical cylinder may not properly represent a composite helix, the bend providing a means for the tertiary structure to assume a globular shape, very much akin to what a proline‐induced kink does to an α‐helix. The tertiary structural context in which β–3₁₀ and 3₁₀–β composites occurs can be different, causing the angle between the secondary structural elements in the two cases to be different. Composites of 3₁₀‐helices and β‐strands are much more conserved among members in families of homologous structures than those between two types of helices; in many of the former instances, the 3₁₀‐helix constitutes the loops in β‐hairpin or β–β‐corner motifs. The overall fold of the chain may be more conserved than the actual identify of the secondary structure elements in a composite. © 2005 Wiley Periodicals, Inc. Biopolymers 78: 147–162, 2005 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structure and conformational stability of a tetrameric thermostable N‐succinylamino acid racemase

The N‐succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co²⁺ by using hydrodynamic and spectroscopic techniques. The Co²⁺, among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by α‐helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten‐globule‐like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co²⁺ did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 757–772, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural analysis of some soluble elastins by means of FT‐IR and 2D IR correlation spectroscopy

Fourier transform infrared (FT‐IR) spectroscopy combined with 2D correlation spectroscopy has been used to offer some information about stability and structure of some soluble elastins. Temperature has been chosen as the perturbation to monitor the infrared behavior of various soluble elastins, namely, α‐elastin p, α‐elastin, and k‐elastin. In the 3800–2700 cm^?1 region, the H‐containing groups were analyzed. The bonded hydroxyls are found to decrease prior to the NH‐related hydrogen bonds and also to the conformational reorganization of hydrocarbon chains. The transition temperatures were evaluated and they were found to agree with those obtained from DSC data. The FTIR spectra and their 2nd derivatives denote that α‐ elastins exhibited amide‐I, ‐II and ‐III bands at 1656, 1539 and 1236 cm^?1, respectively, while in k‐elastin these bands were found at 1652 cm^?1 for amide I, 1540 cm^?1 for amide II and 1248 cm^?1 for amide III. The macroscopic IR finger‐print method, which combines: general IR spectra, secondary derivative spectra, and 2D‐IR correlation spectra, is useful to discriminate different elastins. Thus using the differences of the position and intensity of the bands from “fingerprint region” of studied elastins, which include the peaks assigned to C?O, C? C groups from α‐helix, β‐turn, and the peaks assigned to the amide groups, it is possible to identify and discriminate elastins from each others. Furthermore, the pattern of 2D‐IR correlation spectra under thermal perturbation, allow their direct identification and discrimination. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1072–1084, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The application of DOSY NMR and molecular dynamics simulations to explore the mechanism(s) of micelle binding of antimicrobial peptides containing unnatural amino acids

Anionic and zwitterionic micelles are often used as simple models for the lipids found in bacterial and mammalian cell membranes to investigate antimicrobial peptide‐lipid interactions. In our laboratory we have employed a variety of 1D, 2D, and diffusion ordered (DOSY) NMR experiments to investigate the interactions of antimicrobial peptides containing unnatural amino acids with SDS and DPC micelles. Complete assignment of the proton spectra of these peptides is prohibited by the incorporation of a high percentage of unnatural amino acids which don't contain amide protons into the backbone. However preliminary assignment of the TOCSY spectra of compound 23 in the presence of both micelles indicated multiple conformers are present as a result of binding to these micelles. Chemical Shift Indexing agreed with previously collected CD spectra that indicated on binding to SDS micelles compound 23 adopts a mixture of α‐helical structures and on binding to DPC micelles this peptide adopts a mixture of helical and β‐turn/sheet like structures. DOSY NMR experiments also indicated that the total positive charge and the relative placement of that charge at the N‐terminus or C‐terminus are important in determining the mole fraction of the peptide that will bind to the different micelles. DOSY and ¹H‐NMR experiments indicated that the length of Spacer #1 plays a major role in defining the binding conformation of these analogs with SDS micelles. Results obtained from molecular simulations studies of the binding of compounds 23 and 36 with SDS micelles were consistent with the observed NMR results. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 548–561, 2013.     

Infrared study of synthetic peptide analogues of the calcium‐binding site III of troponin C: The role of helix F of an EF‐hand motif

The EF‐hand motif (helix–loop–helix) is a Ca²⁺‐binding domain that is common among many intracellular Ca²⁺‐binding proteins. We applied Fourier‐transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E–loop–helix F). The 17‐residue peptides corresponding to loop–helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D ‐type amino acid, were investigated to disturb the α‐helical conformation of helix F systematically. These D ‐type‐substituted peptides showed no band at about 1555 cm^?1 even in the Ca²⁺‐loaded state although the native peptide (L ‐type only) showed a band at about 1555 cm^?1 in the Ca²⁺‐loaded state, which is assigned to the side‐chain COO^? group of Glu at the 12th position, serving as the ligand for Ca²⁺ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca²⁺ and the side‐chain COO^? group of Glu at the 12th position. Implications of the COO^? antisymmetric stretch and the amide‐I′ of the synthetic peptide analogues of the Ca²⁺‐binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342–347, 2013.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Glycerol‐induced folding of unstructured disulfide‐deficient lysozyme into a native‐like conformation

2SS[6‐127,64‐80] variant of lysozyme which has two disulfide bridges, Cys6‐Cys127 and Cys64‐Cys80, and lacks the other two disulfide bridges, Cys30‐Cys115 and Cys76‐Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native‐like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4°C. Then, ¹H‐¹⁵N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D₂O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A‐, B‐, and C‐helices and β3‐strand, and especially centered on Ile 55 and Leu 56. In 2SS[6‐127,64‐80], long‐range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 665–675, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com