Human Nucleotide Excision Repair Protein XPA: 1H NMR and CD Solution Studies of a Synthetic Peptide Fragment Corresponding to the Zinc-Binding Domain (101–141)

Abstract

A peptide corresponding to residues 101–141 of the human nucleotide excision repair protein XPA was synthesized with an isoleucine substituted for L138 and its solution structure studied by circular dichroism and homonuclear ¹H NMR spectroscopy. The peptide, (XPA-41), contains a C₄?type zinc-binding motif, C105-(X)₂C108-(X)_l7?C126-(X)₂ C129, which XPA requires for damaged-DNA binding activity. The proton resonances of XPA-41without zinc (apoXPA-41) were assigned using homonuclear TOCSY, NOESY and DQF-COSY data and show the apo-zinc peptide is a random coil. The peptide was folded with the addition of 1.2 equivalents of ZnCl₂ in dilute solution at pH 4.0. Electrospray ionization mass spectroscopy illustrated an increase in the molecular weight of XPA-41 by 65 amu. Circular dichroism spectra of the zinc-folded peptide (zXPA-41) showed the acquisition of elements of secondary structure. Such a conclusion was confirmed with'H NMR data collected at 25°C, pH 6.3. Hα-secondary shifts and NOE patterns indicate that regions V102-C105 and G109-F112 form an anti-parallel β-sheet and residues N128-K137 form a nascent α-helix. Rapid exchange of most amide resonances between S115-C126 prohibited unambiguous assignment of all the proton resonances in this region. However, a 1.19 ppm downfield shift of the H^α resonance of T125 relative to the apo-zinc peptide, together with downfield shifted H^α resonances for the adjacent residues (P124 and L123), suggest a second β-sheet is present in the S115-C126 region. On the basis of structural similarities to GATA-1 (Science 267:438–446), a homology generated structure for zXPA-41 was made, using GATA-1 as the template, which satisfied all the observed NOEs. Using the hybrid homology-NMR based zXPA-41 structure and analogy to GATA-1, models for the role played by the zinc-binding core (101–141) of XPA in DNA damage recognition are proposed.     

A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)₆. Although the binding of tri-N-acetylchitotriose, (GlcNAc)₃, to OEL perturbed several backbone resonances in the ¹H–¹⁵N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pK_a values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)₃ binding. Thermal unfolding experiments and modeling study of (GlcNAc)₆ hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites ?3 and ?2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.     

Differential Cleavage of Urodilatin and Atrial Natriuretic Factor by Thrombin and Protease 3.4.24.11

Abstract

Human urodilatin (residues 95–126) and atrial natriuretic factor (residues 99–126, based on ANF prohor-mone sequence) were incubated separately with three proteases, thrombin, angiotensin converting enzyme (ACE), and neutral endopeptidase 3.4.24.11 (NEP). Thrombin cleaved urodilatin on the carboxyl side of arginine⁹⁸ to yield ANF but under the same conditions did not cleave h-ANF. Neither urodilatin nor ANF was cleaved by ACE. ANF was rapidly degraded by NEP resulting in a major product cleaved between amino acid residues Cys^l05 and Phe¹⁰⁶. Urodilatin was also cleaved by NEP and the amino acid sequencing of the cleaved product revealed the site of cleavage to be the same Cys¹⁰⁵-Phe¹⁰⁶ site as for ANF with a second cleavage site at Gly¹¹⁸-Leu¹¹⁹. However, cleavage of urodilatin by NEP proceeded much more slowly when compared to ANF. A comparison of the affinities of ANF and urodilatin for purified NEP from rabbit kidney revealed K_m values of 11.7 and 3.1 μM, respectively. The turnover rates (k_cat/K_m) for urodilatin and h-ANF with NEP were 4.6 and 37.3 min^?1 μM^?1, respectively. Thus, urodilatin is much less efficiently hydrolyzed by purified NEP than is ANF. The four residue extension at the N-terminus of urodilatin may be important for protection against rapid biological inactivation.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spiderSelenocosmia huwena: 1. Sequence-specific1H-NMR assignments

The complete sequence-specific assignments of resonances in the¹H-NMR spectrum of huwentoxin-I from the Chinese bird spider,Selenocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D₂O and in H₂O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification ofd _N connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification ofd _NN andd _N connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin.Abbreviations HWTX-I huwentoxin-I - 2D two-dimensional - COSY 2D homonuclear correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY 2D nuclear Overhauser enhancement spectroscopy - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP sodium 3-(trimethyl-silyl)propionate-d4 - EDTA ethylenediaminetetraacetic acid     

Competitive peptide antagonists of ANF-induced cyclic guanosine monophosphate production

Atrial natriuretic factor (isoleucine ANF 101-126), cleaved ANF (isoleucine ANF 101-105/106-126) and des (Gln 18, Ser 19, Gly 20, Leu 21, Gly 22) ANF 4-23-NH2 (C-ANF 4-23) stimulated cyclic guanosine monophosphate production (cGMP) by rat aortic vascular smooth muscle cells (VSMC) in culture. Cleaved ANF and ANF C4-23 also antagonised or diminished the response to ANF 101-126. Agonist and antagonist actions of both peptides were dose-related. In contrast, prepro ANF (104-123), an ANF precursor fragment, exhibited no agonist or antagonist effect on cGMP production.     

Bivariate assay for cleaved atrial natriuretic factor (ANF(101–105/106–126)) in sheep plasma

A bivariate assay has been developed to measure both ANF, and cleaved ANF (ANF(101–105/106–126)) the primary product of endopeptidase-24.11 action on rat ANF(101–126). The assay employed two different antisera directed to the carboxy-terminal and ring regions of ANF to enable discrimination and measurement of intact endogenous ANF(99–126) and infused cleaved ANF (rat ANF(101–105/106–126)) in sheep plasma extracts. The assay which was validated by HPLC analysis of plasma extracts, had sufficient accuracy and precision to measure the metabolic clearance rate and half-life of cleaved ANF when infused in normal sheep.     

Solution structure of human calcitonin in membrane-mimetic environment: The role of the amphipathic helix

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), ³J_HNα coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic α-helix from Leu9 to Phe16, followed by a type-I β-turn between residues Phe16 and Phe19. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure–activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed. Proteins 32:314–323, 1998. © 1998 Wiley-Liss, Inc.     

Neiella holothuriorum sp. nov., isolated from the gut of a sea cucumber Apostichopus japonicus

A Gram-stain negative, aerobic, rod-shaped bacterium, designated 126^T, was isolated from the intestinal content of a sea cucumber, Apostichopus japonicus, in China. Strain 126^T was found to grow optimally at 25–28 °C and pH 7.5–8.0 in marine 2216 E medium, with tolerance of 1–7% (w/v) NaCl. Strain 126^T is motile by means of one to several polar flagella. The dominant fatty acids of strain 126^T were identified as C_16:1 ω7c/C_16:1 ω6c (29.5%), C_18:1 ω7c/C_18:1 ω6c (19.8%) and C_16:0 (16.7%). The respiratory quinone was found to be Q-8. The polar lipid profile was found to be mainly composed of phosphatidylglycerol and phosphatidylethanolamine. The total length of the draft genome is approximately 4.2?×?10⁶ bp, encoding 3655 genes and 3576 coding sequences. The G?+?C content of the genomic DNA is 48.0%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 126^T belongs to the genus Neiella and is closely related to Neiella marina J221^T (96.5%). Genomic comparisons of 126^T to N. marina J221^T revealed that they had similar genome size, G?+?C content and complement of clusters of orthologous groups. However, average nucleotide identity and digital DNA–DNA hybridization values between strains126^T and N. marina J221^T was 75.5% and 19.7%, which could distinguish the strains. On the basis of these phenotypic and genotypic data, strain 126^T is concluded to represent a novel species, for which the name Neiella holothuriorum sp. nov. is proposed. The type strain is 126^T (=?GDMCC 1.2530^T?=?KCTC 82829^T).

     

Conformational differences between cis and trans proline isomers of a peptide antigen representing the receptor binding domain of Pseudomonas aeruginosa as studied by 1H-nmr

A 17-residue disulfide-bridged peptide (PAK 128–144) corresponding to the C-terminus of Pseudomonas aeruginosa pilin strain K has been studied by one- and two-dimensional nmr techniques. This synthetic immunogen has been found to exist as two distinct conformations in solution, which have been demonstrated to arise as a result of the isomerization of the I¹³⁸-P¹³⁹ amide bond. The two isomers occur in the ratio of 3 : 1 trans to cis at 5°C. Sequential assignments for both forms have been accomplished through the use of nuclear Overhauser enhancement spectroscopy (NOESY) spectra and most side-chain resonances have been assigned using a combination of correlated spectroscopy, total correlated spectroscopy, and NOESY spectra. The presence of the cis isomer, which is considerably more predominant in the oxidized peptide, was confirmed by the observation of the characteristic NOEs between P¹³⁹ and the preceding residue. Further corroboration was given by the disappearance of the cis resonances in the spectrum of the P139A analogue of PAK 128–144. From observation of the differences in the chemical shifts and amide proton temperature coefficients of the two isomers, it is apparent that the two forms differ markedly in their solution conformation. The biological implications of the isomerization are discussed. © 1994 John Wiley & Sons, Inc.     

Radioautographic localization of125I-atrial natriuretic fator (ANF) in rat tissues

Summary Rats were injected either with synthetic¹²⁵I-Arg 101-Tyr 126 atrial natriuretic factor (ANF) or with¹²⁵I-ANF together with an excess of cold Arg 101-Tyr 126 ANF. Binding sites in various tissues were accepted depending on two criteria: displacement of radioactivity by cold ANF and absence of localization of silver grains on putative target cells in the presence of cold ANF. Binding sites were localized on zona glomerulosa cells and on adrenergic and noradrenergic cells of adrenal medulla, on hepatocytes, on the base of mature epithelial cells of villi in the small intestine, on smooth muscle cells of the muscularis layer of the colon and on the base of epithelial cells of the ciliary bodies. In addition, binding sites were localized in the vasculature of kidney, adrenal cortex, lung and liver. Binding sites were particularly numerous on renal glomerular endothelial cells. These results indicate that ANF may have important hemodynamic effects in kidney, lung, liver and adrenal cortex, may regulate water and ion transport in small intestine and ciliary bodies and may have metabolic effects in the liver. The presence of binding sites on the zona glomerulosa is in agreement with the important inhibitory effect of the peptide on aldosterone secretion.     

Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: Relationship to expression of ANF-C receptors

Previous studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C-type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum-stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF_99–126, rANF_103–126, and rANF_103–125, were only poorly antimitogenic in serum-stimulated primary cultures, whereas des[Cys¹⁰⁵, Cys¹²¹] rANF_104–126 which binds selectively to the ANF-C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF_99–126, rANF_103–126, rANF_103–126, Cys¹¹⁶rANF_102–116, and des[Cys¹⁰⁵, Cys¹²¹]rANF_104–126 inhibited serum-induced [³H]thymidine incorporation (IC₅₀ in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP-elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric-oxide-vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [¹²⁵I]rANF_99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF-C-type type receptor. Covalent cross-linking studies with (¹²⁵I)rANF_99–126 confirmed that membranes prepared from fresh aortae predominantly expressed the ANF-A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross-linked protein was the ANF-C-type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased expression of the ANF-C-type receptors. © 1993 Wiley-Liss, Inc.     

Modulation of the electron-proton coupling at cytochrome a by the ligation of the oxidized catalytic center in bovine cytochrome c oxidase

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a₃ (Fe_a3) and copper (Cu_B). Here, the influence of the ligation at the oxidized Fe_a3³⁺–Cu_B²⁺ center on the electron–proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (E_m(a)) for the cyanide (the low-spin Fe_a3³⁺) and the formate-ligated CcO (the high-spin Fe_a3³⁺). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of E_m(a) were found for the CN– and the formate–ligated CcO with slopes of −13 mV/pH unit and −23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron–proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron–proton coupling at the physiological pH values is also substantiated by the UV–Vis absorption and electron–paramagnetic resonance spectroscopy investigations of the cyanide–ligated oxidized CcO. It is shown that the ligand exchange at Fe_a³⁺ between His–Fe_a³⁺–His and His–Fe_a³⁺–OH⁻ occurs only at pH above 9.5 with the estimated pK >11.0.     

Reliability of exclusively NOESY-based automated resonance assignment and structure determination of proteins

Protein structure determination by NMR can in principle be speeded up both by reducing the measurement time on the NMR spectrometer and by a more efficient analysis of the spectra. Here we study the reliability of protein structure determination based on a single type of spectra, namely nuclear Overhauser effect spectroscopy (NOESY), using a fully automated procedure for the sequence-specific resonance assignment with the recently introduced FLYA algorithm, followed by combined automated NOE distance restraint assignment and structure calculation with CYANA. This NOESY-FLYA method was applied to eight proteins with 63–160 residues for which resonance assignments and solution structures had previously been determined by the Northeast Structural Genomics Consortium (NESG), and unrefined and refined NOESY data sets have been made available for the Critical Assessment of Automated Structure Determination of Proteins by NMR project. Using only peak lists from three-dimensional ¹³C- or ¹⁵N-resolved NOESY spectra as input, the FLYA algorithm yielded for the eight proteins 91–98 % correct backbone and side-chain assignments if manually refined peak lists are used, and 64–96 % correct assignments based on raw peak lists. Subsequent structure calculations with CYANA then produced structures with root-mean-square deviation (RMSD) values to the manually determined reference structures of 0.8–2.0 Å if refined peak lists are used. With raw peak lists, calculations for 4 proteins converged resulting in RMSDs to the reference structure of 0.8–2.8 Å, whereas no convergence was obtained for the four other proteins (two of which did already not converge with the correct manual resonance assignments given as input). These results show that, given high-quality experimental NOESY peak lists, the chemical shift assignments can be uncovered, without any recourse to traditional through-bond type assignment experiments, to an extent that is sufficient for calculating accurate three-dimensional structures.     

Localization of binding sites for atrial natriuretic factor and angiotensin II in the central nervous system of the clawed toad Xenopus laevis

Summary The distribution of binding sites for atrial natriuretic factor (ANF) and angiotensin II (A II) was investigated in the central nervous system (CNS) of the clawed toad Xenopus laevis by means of in vitro autoradiography using [¹²⁵I]-rat ANF(99–126) or [¹²⁵I] [Val⁵] A II and [¹²⁵I]human A II as labeled ligands. The highest densities of specific ANF-binding were detected in the nucleus habenularis, thalamic regions, hypophyseal pars nervosa and nucleus interpeduncularis. Moderate ANF-binding was found in the bulbus olfactorius, pallium, septum, striatum, lateral forebrain bundle, nucleus infundibularis, hypophyseal pars distalis and tectum. The highest levels of specific A II binding sites were observed in the nucleus praeopticus, nucleus habenularis, hypophyseal pars nervosa and pars distalis, whereas the amygdala contained moderate A II binding. The existence of specific binding sites for ANF and A II in the CNS of Xenopus laevis suggests that both peptides act as neurotransmitters or neuromodulators in the amphibian CNS. The co-localization of dense binding sites for both peptides in the nucleus habenularis, hypophyseal pars nervosa and pars distalis supports the view that ANF and A II have opposite regulatory functions in these regions.     

Effect of ante- and postmortem hide clipping on the microbiological quality and safety and ultimate pH value of beef carcasses in an EC-approved abattoir

Aims:  Effect of ante- and postmortem hide clipping on the microbiological quality of beef carcasses. Methods and Results:  Bovine carcasses (362) were tested for indicator micro-organisms and the presence of pathogens. Prior to slaughter, hide cleanliness of each animal was categorized on a scale of 1–5 (clean to dirty). Lowest mean aerobic colony counts (ACC) (log₁₀ 3·0 CFU cm⁻²) came from carcasses where clipping had been performed in lairage, antemortem. ACC from animals clipped online (log₁₀ 3·2 CFU cm⁻²) were significantly higher (P < 0·05) than those clipped in lairage, but comparable to those carcasses from Category 1 and 2 animals. There were no significant differences in the detection of pathogens from any of the carcass groups. Ultimate pH values for carcasses from Category 3 and 4 animals showed clipping animals in lairage, as opposed to online, resulted in a small, but significant increase (P < 0·05) in pH value (mean pH 5·66 and 5·59, respectively). Conclusions:  Hide clipping does not adversely affect microbiological quality of carcasses, although higher ultimate pH values indicate increases in antemortem stress. Significance and Impact of the Study:  Hide clipping carcasses both ante- and postmortem appears to be an effective intervention to minimize transfer of hide microflora to carcasses during slaughtering operations. Online clipping offers advantages for animal welfare and improves safety for operatives.     

The random coil leads to beta transition of copolymers of L-lysine and L-valine: potentiometric titration and circular dichroism studies.

A series of copolymers of L -lysine and L -valine [poly(L -lysine^f L -valine^100-f)] containing 0–13% L -valine have been studied, in 0.10M KF solution, using potentiometric titration and circular dichroism spectroscopy. Incorporation of increasing amounts of valine into the copolymers favors β-sheet formation over α-helix formation at high pH and room temperature. The titrations were analyzed using the method of Zimm and Rice and the partial free energy (ΔG⁰_cβ) for the coil-to-β-sheet transition for valine is estimated at 900 cal/mole at 25°C. From the temperature dependence of the free energy, the partial enthalpy, ΔH⁰_cβ, and entropy, ΔS⁰_cβ, of the transition for valine is estimated to be 854 cal/mole and 6.0 e.u., respectively. The corresponding partial thermodynamic parameters for L -lysine are in agreement with published results. The fraction of β-sheet versus pH has been calculated for poly(L -lysine^86.8 L -valine^13.2) at 25.0°C using the titration data; data obtained from circular dichroism spectroscopy for the same copolymer are in good accord. It is concluded from these results that L -valine is a very strong β-sheet forming amino acid. Furthermore, these results indicate that the Zimm–Rice method is applicable to transitions between the coil and β-sheet states for a polypeptide containing two different residues.     

Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

Summary The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50% H₂O/CD₃OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The H ^N -H ^N sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The α-proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H ₂ ^α -H ^NEth is indicative of proximity between C- and N-termini.     

Resonance energy transfer,pH‐induced folded states and the molecular interaction of human serum albumin and icariin

Icariin is a flavonol glycoside with a wide range of pharmacological and biological activities. The pharmacological and biological functions of flavonoid compounds mainly originate from their binding to proteins. The mode of interaction of icariin with human serum albumin (HSA) has been characterized by fluorescence spectroscopy and far‐ and near‐UV circular dichroism (CD) spectroscopy under different pH conditions. Fluorescence quenching studies showed that the binding affinity of icariin with HSA in the buffer solution at different pH values is: K_a (pH 4.5) > K_a (pH 3.5) > K_a (pH 9.0) > K_a (pH 7.0). Red‐edge excitation shift (REES) studies revealed that pH had an obvious effect on the mobility of the tryptophan microenvironment and the addition of icariin made the REES effect more distinct. The static quenching mechanism and number of binding sites (n ≈ 1) were obtained from fluorescence data at three temperatures (298, 304 and 310 K). Both ?H⁰ < 0 and ??⁰ < 0 suggested that hydrogen bonding and van der Waal's interaction were major driving forces in the binding mechanism, and this was also confirmed by the molecular simulation results. The distance r between the donor (HSA) and the acceptor (icariin) was calculated based on Förster non‐radiation energy transfer theory. We found that pH had little impact on the energy transfer between HSA and icariin. Far‐ and near‐UV CD spectroscopy studies further indicated the influence of pH on the complexation process and the alteration in the protein conformation upon binding. Copyright © 2015 John Wiley & Sons, Ltd.