Amide Proton Temperature Coefficients Research Articles

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis

Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional 19F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional 19F spectra to those from two-dimensional 1H-15N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional 19F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of 19F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis.

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature‐dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics. Fundamentally, these temperature dependences result from changes in the local magnetic environments of nuclei, but correlations with global thermodynamic parameters measured via calorimetric methods have been observed. Although the temperature dependences of amide proton and nitrogen chemical shifts are often well approximated by a linear model, deviations from linearity are also observed and may be interpreted as evidence of fast exchange between distinct conformational states. Here, we describe computational methods, accessible via the Shift‐T web server, including an automated tracking algorithm that propagates initial (single temperature) 1H—15N cross peak assignments to spectra collected over a range of temperatures. Amide proton and nitrogen temperature coefficients (slopes determined by fitting chemical shift vs. temperature data to a linear model) are subsequently calculated. Also included are methods for the detection of systematic, statistically significant deviation from linearity (curvature) in the temperature dependences of amide proton chemical shifts. The use and utility of these methods are illustrated by example, and the Shift‐T web server is freely available at http://meieringlab.uwaterloo.ca/shiftt.

Investigation of the Amide Proton Solvent Exchange Properties of Glycosaminoglycan Oligosaccharides.

One-dimensional 1H NMR experiments were conducted for aqueous solutions of glycosaminoglycan oligosaccharides to measure the amide proton temperature coefficients and activation energy barriers for solvent exchange and evaluate the effect of pH on the solvent exchange properties. A library of mono- and oligosaccharides was prepared by enzymatic depolymerization of amide-containing polysaccharides and by chemical modification of heparin and heparan sulfate saccharides including members that contain a 3- O-sulfated glucosamine residue. The systematic evaluation of this saccharide library facilitated assessment of the effects of structural characteristics, such as size, sulfation number and site, and glycosidic linkage, on amide proton solvent exchange rates. Charge repulsion by neighboring negatively charged sulfate and carboxylate groups was found to have a significant impact on the catalysis of amide proton solvent exchange by hydroxide. This observation leads to the conclusion that solvent exchange rates must be interpreted within the context of a given chemical environment. On their own, slow exchange rates do not conclusively establish the involvement of a labile proton in a hydrogen bond, and additional supporting experimental evidence such as reduced temperature coefficients is required.

Structural Determination of the Tandem Repeat Motif in Samia cynthia ricini Liquid Silk by Solution NMR

The wild silkworm Samia cynthia ricini produces silk fibroin containing polyalanine sequences. The structure of a typical tandem repeat sequence YGGDGG(A)12GGAG within S. c. ricini silk fibroin in liquid silk was determined by solution NMR. 13C, 15N, and 1H shifts were assigned from solution NMR spectra for the tandem repeat. TALOS-N was then used to predict the backbone dihedral angles from the chemical shifts. Dihedral angles revealed a well-structured α-helix for the polyalanine region and less stable capping motifs for the N- and C-terminal regions. Consistent with this, the amide proton temperature coefficients of the last glycine in the N-terminal region and the first two glycines in the C-terminal region show more positive values than that of a random coil structure. Thus, the α-helical structure of the polyalanine region of S. c. ricini liquid silk is stabilized by capping motifs.

β-Hairpin Peptide That Targets Vascular Endothelial Growth Factor (VEGF) Receptors

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.

Temperature dependencies of amide 1H‐ and 15N‐chemical shifts in hyaluronan oligosaccharides

Temperature coefficients (Deltadelta/DeltaT) of amide chemical shifts of N-acetylglucosamine residues have been measured in a range of oligosaccharides of the important vertebrate polysaccharide hyaluronan. Odd- and even-numbered oligosaccharides with glucuronic acid, Delta-4,5-unsaturated glucuronic acid and N-acetylglucosamine at the termini were investigated. All amide proton temperature coefficients were only slightly less negative (-6.9 to - 9.1 ppb/ degrees C) than those of amide protons in free exchange with water (approximately equal to -11 ppb/ degrees C), indicating an absence of persistent intramolecular hydrogen bonds. With the exception of amide groups in reducing-terminal N-acetylglucosamine rings, all amide proton environments have the same temperature coefficient (-6.9 ppb/ degrees C), irrespective of differences in amide group chemical shifts and (3)J(HH) coupling constants, i.e. they do not sense subtle differences in orientation of the amide group. Amide nitrogen temperature coefficients report the same phenomena but with greater sensitivity. These data provide a set of reference values for temperature coefficients measured in other carbohydrates with acetamido sugars.

NMR analysis of G7‐18NATE, a nonphosphorylated cyclic peptide inhibitor of the Grb7 adapter protein

G7-18NATE is a nonphosphorylated, cyclic peptide that specifically inhibits the Grb7 adapter protein implicated in several pathways critical to cell proliferation and migration. It has been shown that G7-18NATE is able to compete with natural ligands for the Grb7 SH2 phosphotyrosine binding site, and to attenuate cell migration in a pancreatic cancer cell line. It is thus an important lead in the development of a selective inhibitor of Grb7 and potential novel anticancer therapeutics. The current study reports the solution properties of G7- 18NATE determined using NMR spectroscopy, in both water (pH 2-3) and phosphate buffer (pH 6.0), with 100 mM NaCl. The spectra reveal that G7-18NATE exists in two distinguishable conformational states on the NMR timescale, most likely due to cis-trans proline isomerization. In addition, the chemical shift data are consistent with a tendency of G7-18NATE to form a turn about the YDN motif, known to be important for binding, and suggest that this turn is stabilized in low salt and low pH conditions. Low NH temperature coefficients of Tyr-5 and Asn-7 amide protons may reflect their involvement in the formation of hydrogen bonds that stabilize such a turn. Overall, however, the peptide does not form a rigid structure, but exists in a highly flexible state in solution. Averaged 3JNH-H coupling constants and a lack of interresidue NOEs are characteristic of such peptide solution behavior. This suggests that there is scope for increasing the rigidity of the peptide that may enhance its binding affinity and specificity for Grb7.

The β-turn scaffold of tripeptide containing an azaphenylalanine residue

The conformational preferences of azaphenylalanine-containing peptide were investigated using a model compound, Ac-azaPhe-NHMe with ab initio method at the HF/3-21G and HF/6-31G ⁎ levels, and the seven minimum energy conformations with trans orientation of acetyl group and the 4 minimum energy conformations with cis orientation of acetyl group were found at the HF/6-31G ⁎ level if their mirror images were not considered. An average backbone dihedral angle of the 11 minimum energy conformations is ϕ = ± 91° ± 24°, ψ = ± 18° ± 10° (or ± 169° ± 8°), corresponding to the i + 2 position of β-turn ( δ R) or polyproline II ( β P) structure, respectively. The χ 1 angle in the aromatic side chain of azaPhe residue adopts preferentially between ± 60° and ± 130°, which reflect a steric hindrance between the N-terminal carbonyl group or the C-terminal amide group and the aromatic side chain with respect to the configuration of the acetyl group. These conformational preferences of Ac-azaPhe-NHMe predicted theoretically were compared with those of For-Phe-NHMe to characterize the structural role of azaPhe residue. Four tripeptides containing azaPhe residue, Boc-Xaa-azaPhe-Ala-OMe [Xaa = Gly( 1), Ala( 2), Phe( 3), Asn( 4)] were designed and synthesized to verify whether the backbone torsion angles of azaPhe reside are still the same as compared with theoretical conformations and how the preceding amino acids of azaPhe residue perturb the β-turn skeleton in solution. The solution conformations of these tripeptide models containing azaPhe residue were determined in CDCl 3 and DMSO solvents using NMR and molecular modeling techniques. The characteristic NOE patterns, the temperature coefficients of amide protons and small solvent accessibility for the azapeptides 1–4 reveal to adopt the β-turn structure. The structures of azapeptides containing azaPhe residue from a restrained molecular dynamics simulation indicated that average dihedral angles [( ϕ 1, ψ 1), ( ϕ 2, ψ 2)] of Xaa-azaPhe fragment in azapeptide, Boc-Xaa-azaPhe-Ala-OMe were [(− 68°, 135°), (116°, − 1°)], and this implies that the intercalation of an azaPhe residue in tripeptide induces the βII-turn conformation, and the volume change of a preceding amino acid of azaPhe residue in tripeptides would not perturb seriously the backbone dihedral angle of β-turn conformation. We believe such information could be critical in designing useful molecules containing azaPhe residue for drug discovery and peptide engineering.

Probing the folding capacity and residual structures in 1–79 residues fragment of staphylococcal nuclease by biophysical and NMR methods

1–79 residues SNase fragment (SNase79) has chain length containing a sequence for helix α 1, ω-loop, β I-sheet, and partial β II-sheet of native SNase. The incomplete “β-barrel” structural region of SNase79 makes this fragment to be interested in investigation of its conformation. For this study, we use CD, fluorescence, and NMR spectroscopy to probe the folding capacity and the residual structures in SNase79. The optical spectra obtained for SNase79 and its mutants reveal the presence of retained capacity for folding of the fragment. The NMR derived 13C α secondary chemical shifts, 3 J NH–Hα coupling constants, amide-proton temperature coefficients, interresidue NOEs, and 15N relaxation data determine the intrinsic propensities for helix- and turn- or β-sheet-like conformations of SNase79, which is not the result of stabilizing inter-molecular interactions by oligomerization effects. The residual turn- and helix-like structures may serve as potential local nucleation sites, whereas the residual β I-sheet-like structure can be regarded as a potential non-local nucleation site in the folding of SNase79. The intrinsic local and non-local interactions in these potential initiation sites are insufficient to stabilize the folding of SNase79 due to the shortage of relevant long-range interactions from other part of the fragment. The conformational ensemble of SNase79 is a highly heterogeneous collection of interconverting conformations having transiently populated helix- and β-sheet- or turn-like structures.

Hyaluronan: the absence of amide-carboxylate hydrogen bonds and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure models.

Contradictory descriptions for the aqueous solution conformation of the glycosaminoglycan hyaluronan (HA) exist in the literature. According to hydrodynamic and simulation data, HA molecules are stiffened by a rapidly interchanging network of transient hydrogen bonds at the local level and do not significantly associate at the global level. In marked contrast, models derived from NMR data suggest that the secondary structure involves persistent hydrogen bonds and that strong associations between chains can occur to form vast stable tertiary structures. These models require an extended 2-fold helical conformation of the HA chain and specific hydrogen bonds between amide and carboxylate groups. To test these descriptions, we have used 15N-labelled oligosaccharides and high-field NMR to measure pertinent properties of the acetamido group. The amide proton chemical shift perturbation and carboxylate group pK(a) value are inconsistent with a highly populated hydrogen bond between the amide and carboxylate groups. Amide proton temperature coefficients and chemical exchange rates confirm this conclusion. Comparison of oligomer properties with polymeric HA indicates that there is no discernible difference in amide proton environment between the centre of octasaccharides and the polymer, inconsistent with the formation of tertiary structures. A [1H-1H-15N] NOESY-HSQC (heteronuclear single-quantum correlation) spectrum recorded on an HA octasaccharide revealed that amide groups in the centre are in a trans orientation and that the average solution conformation is not an extended 2-fold helix. Therefore the two key aspects of the secondary and tertiary structure models are unlikely to be correct. Rather, these new NMR data agree with descriptions from hydrodynamic and simulations data.

Structural and Dynamic Characterization of the Acid-Unfolded State of hUBF HMG Box 1 Provides Clues for the Early Events in Protein Folding,

To understand the events that occur in the early stages of the folding of hUBF HMG box 1, we characterized its pH 2.1 unfolded state in detail with NMR. Through a triple resonance strategy, the assignments of complete backbone and some side chains were achieved. Then, significant conformational information was extracted from secondary chemical shifts, interresidual (1)H-(1)H NOEs, (3)J(HNHA) coupling constants, amide proton temperature coefficients, and (15)N relaxation data. The secondary chemical shifts for (13)CA, (13)CB, (13)CO, (1)HA, and (1)HN indicate that the residues between 64 and 78 exhibit a substantial preference for helical structure in the acid-unfolded state, which is also evidenced by the relatively more negative deviations of (3)J(HNHA) and amide proton temperature coefficients from their corresponding random-coil values and particularly confirmed by the strongest sequential d(NN)(i, i + 1) proton NOEs along the region. Following this region until residue 82 is a segment that tends to form a turn-like structure, which is unstable and exchanges between alternative states. In addition, some evidences imply that the regions 18-28 and 38-43 also possess propensities for helical structure but to a different less degree than the region 64-78. The polypeptide backbone dynamics investigated using reduced spectral density function shows apparent motional restrictions in residual structural regions and to less extent at some hydrophobic residues. On the basis of the results presented herein, we propose a potential protein-folding pathway on which these residual structures play a role of initiation site in the early folding stages.

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, raman, CD spectroscopy and molecular modeling

The synthesis and biological evaluation of penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs and comparison with their proline(7)-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen(1),Pen(6)]-oxytocin (1, pA(2) = 8.22, EC(50) = 6.0 nM) and [dPen(1),Pen(6),5-tBuPro(7)]-oxytocin (2, pA(2) = 8.19, EC(50) = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using (1)H NMR, laser Raman and CD techniques. In H(2)O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln(4) and Pen(6) in the major trans-conformer of 1 as well as for Gln(4) in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 +/- 0.2 kcal/mol) for isomerization of the amide N-terminal to 5-tBuPro(7) relative to OT was calculated from measuring the coalescence temperature of the Gly(9) backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C(beta)-S-S-C(beta) dihedral angles of +110-115 degrees and +/-90 degrees , respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a chi(CS-SC) dihedral angle value close to 115 degrees was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a +/-90 degrees angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a +/-90 degrees chi(CS-SC) dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114 degrees and +116 degrees . Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104 degrees . In sum, the conformation about the disulfide in [dPen(1),Pen(6)]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen(1),Pen(6),5-tBuPro(7)]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro(7) (5R)-tert-butyl group, the Leu(8) isopropyl sidechain and the Pen(6)beta-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110 degrees may be a prerequisite for their binding at the receptor.

Hydrogen bonding on the ice-binding face of a beta-helical antifreeze protein indicated by amide proton NMR chemical shifts.

The dependence of amide proton chemical shifts on temperature is used as an indication of the hydrogen bonding properties in a protein. The amide proton temperature coefficients of the beta-helical antifreeze protein from Tenebrio molitor are examined to determine their hydrogen bonding state in solution. The temperature-dependent chemical shift behavior of the amides in T. molitor antifreeze protein varies widely throughout the protein backbone; however, very subtle effects of hydrogen bonding can be distinguished using a plot of chemical shift deviation (CSD) versus the backbone amide chemical shift temperature gradient (Deltadelta/DeltaT). We show that differences between the two ranks of ice-binding threonine residues on the surface of the protein indicate that threonine residues in the left-hand rank participate in intrastrand hydrogen bonds that stabilize the flat surface required for optimal ice binding.

NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor, hGRF(1-29)-NH2.

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.

Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange.

The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding.

1H NMR studies on the solution conformation of contulakin-G and analogues

The conformation of contulakin-G, a bioactive 16 amino acid O-linked glycopeptide (ZSEEGGSNAT*KKPYIL) with the disaccharide β-D-Gal(1[Formula: see text]3)α-D-GalNAc attached to the threonine residue in position 10, has been investigated by 1H NMR spectroscopy. The 1H NMR data for the non-glycosylated peptide and for two glycopeptide analogues, one with the monosaccharide α-D-GalNAc at Thr10 and one with the disaccharide β-D-Gal(1–>3)α-D-GalNAc at Ser7, all of lower bioactivity than contulakin-G, have also been collected. The chemical shifts, NOEs, temperature coefficients of amide protons, and 3JNH,αH-values suggest that all four compounds exist mainly in random coil conformations. Some transient populations of folded conformations are also present in the glycopeptides and turns, probably induced by the sugars, are present in the peptide chain around the site of glycosylation. In the two peptides O-glycosylated at Thr10, the rotation of α-D-GalNAc around the linkage between the sugar and the peptide is restricted. There is evidence for a hydrogen bond between the amide proton of α-D-GalNAc and the peptide chain that could contribute to this torsional rigidity. An intramolecular hydrogen bond between the carbohydrate and the peptide chain does not exist in the peptide O-glycosylated at the Ser7 residue. Key words: conformation, contulakin-G, NMR, O-linked glycopeptide.

Hydrogen Bonds in Human Ubiquitin Reflected in Temperature Coefficients of Amide Protons

Analysis of amide proton temperature coefficients (Δσ HN/Δ T) in human ubiquitin shows their usefulness in indicating hydrogen bonds. The availability of a very accurate solution structure of ubiquitin enables the precise determination of hydrogen bonds and increases the reliability of the analysis of chemical shift temperature gradients. Values of Δσ HN/Δ T more positive than −4.6 ppb/K are very good indicators of hydrogen bonds. Additionally, a weak temperature dependence of non-hydrogen-bonded amides was observed for amide protons that are significantly shifted upfield. We observed that temperature gradients of amide protons involved in short hydrogen bonds are related to donor–acceptor distances.

Mapping the targeted membrane pore formation mechanism by solution NMR: the nisin Z and lipid II interaction in SDS micelles.

Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings and unusual dehydrated amino acids. Among the numerous pore-forming antimicrobial peptides, type-A lantibiotics form an unique family of post-translationally modified peptides. Via the recognition of cell wall precursor lipid II, nisin has the capacity to form pores against Gram-positive bacteria with an extremely high activity in the nanomolar (nM) range. Here we report a high-resolution NMR spectroscopy study of nisin/lipid II interactions in SDS micelles as a model membrane system in order to elucidate the mechanism of molecular recognition at residue level. The binding to lipid II was studied through (15)N-(1)H HSQC titration, backbone amide proton temperature coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics experiments. Upon the addition of lipid II, significant changes were monitored in the N-terminal part of nisin. An extremely low amide proton temperature coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (> -0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or isolation from the bulk solvent for this residue. Large chemical shift perturbations were also observed in the first two rings. In contrast, the C-terminal part of nisin was almost unaffected. This part of the molecule remains flexible and solvent-exposed. On the basis of our results, a multistep pore-forming mechanism is proposed. The N-terminal part of nisin first binds to lipid II, and a subsequent structural rearrangement takes place. The C-terminal part of nisin is possibly responsible for the activation of the pore formation. In light of the emerging antibiotic resistance problems, an understanding of the specific recognition mechanism of nisin with lipid II at the residue specific level may therefore aid in the development of novel antibiotics.

Effect of sequence on peptide geometry in 5-tert-butylprolyl type VI beta-turn mimics.

The influence of sequence on turn geometry was examined by incorporating (2S,5R)-5-tert-butylproline (5-(t)BuPro) into a series of dipeptides and tetrapeptides. (2S,5R)-5-tert-Butylproline and proline were respectively introduced at the C-terminal residue of N-acetyl dipeptide N'-methylamides 1 and 2. The conformational analysis of these analogues was performed using NMR and CD spectroscopy as well as X-ray diffraction to examine the factors that control the prolyl amide (in this text, the term "prolyl amide" refers to the tertiary amide composed of the pyrrolidine nitrogen of the prolyl residue and the carbonyl of the N-terminal residue) equilibrium and stabilize type VI beta-turn conformation. The high cis-isomer population with aromatic residues N-terminal to proline was shown to result from a stacking interaction between the partial positive charged prolyl amide nitrogen and the aromatic pi-system as seen in the crystal structure of 1c. The effect of sequence on the prolyl amide equilibrium of 5-(t)BuPro-tetrapeptides (Ac-Xaa-Yaa-5-(t)BuPro-Zaa-XMe, 13 and 14) was studied by varying the amino acids at the Xaa, Yaa, and Zaa positions. High (>80%) cis-isomer populations were obtained with alkyl groups at the Xaa position, an aromatic residue at the Yaa position, and either an alanine or a lysine residue at the Zaa position of the 5-(t)BuPro-tetrapeptide methyl esters in water. Tetrapeptides Ac-Ala-Phe-5-(t)BuPro-Zaa-OMe (Zaa = Ala, Lys), 14d and 14f, with high cis-isomer content adopted type VIa beta-turn conformations as shown by their NMR and CD spectra. Although a pattern of amide proton temperature coefficient values indicative of a hairpin geometry was observed in peptides 14d and 14f, the value magnitudes did not indicate strong hydrogen bonding in water.

NMR identification of local structural preferences in HIV-1 protease tethered heterodimer in 6 M guanidine hydrochloride.

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus-1 protease in the 'unfolded state'. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native-like, and there are also some non-native structural elements. Three-bond H(N)-H(alpha) coupling constants that could be measured for some of the N-terminal and C-terminal residues are consistent with the native-like beta-structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.