Deuterium NMR of 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A in oriented DMPC bilayers.

Deuterium NMR is used to study the structure and dynamics of the formyl C-2H bond in selectively deuterated gramicidin molecules. Specifically, the functionally different analogues 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A are studied by 2H NMR so that any conformational or dynamical differences between the two analogues can be correlated with their difference in lifetime. These analogues are first synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorous NMR line shapes obtained from these samples are consistent with the presence of the bilayer phase and indicate that the disorder exhibited by the lipid matrix is approximately of the same type and degree for both analogues. Deuterium NMR line shapes obtained from these samples indicate that the motional axis of the formyl group of gramicidin is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 7-9 degrees, and that a similar, major conformational and dynamical state exists for the formyl C-2H bond of both analogues. In this state, if the only motion present is fast axial rotation, then the experimentally derived angle between the formyl C-2H bond and the motional axis is consistent with the presence of a right-handed, single-stranded, beta 6.3 helical dimer but is not consistent with the presence of a left-handed, single-stranded, beta 6.3 helical dimer. However, if fast axial rotation is not the only motion present, then the left-handed, single-stranded, beta 6.3 helical dimer cannot be absolutely excluded as a possibility. Also, a second, minor conformational and dynamical state appears to be present in the spectrum of 2HCO-Val1-D-Leu2...gramicidin A but is not observed in the spectrum of 2HCO-Val1...gramicidin A. This minor conformational and dynamical state may reflect the presence of monomers, while the major conformational and dynamical state may reflect the presence of dimers.     

2H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer.

Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there.     

Orientation of the valine-1 side chain of the gramicidin transmembrane channel and implications for channel functioning. A 2H NMR study.

The orientation of the valine-1 side chain of gramicidin was determined by solid-state 2H NMR using valine-1-deuterated (d8) gramicidin. The peptide was incorporated into DMPC bilayers that were oriented between glass plates. When the plates were oriented with their normal perpendicular to the magnetic field, four quadrupolar splittings were observed of 106, 68, 9.7, and 2.0 kHz. These resonances were assigned to C alpha D, C beta D, and the deuterons of each of the C gamma D3 methyl groups, respectively. The average orientation of the various C-D bonds was calculated with respect to the helix axis. The angle obtained for the C alpha-D resonance was consistent with a single-stranded beta 6.3-helical model for the backbone but not with double-helical models. The angles of the side chain were then fitted to a model for the right-handed beta 6.3-helix. Rotation of the valine-1 side chain yielded a set of torsion angles that matched the angles as determined from the 2H NMR measurements. The corresponding orientation of the valine-1 side chain (chi 1 = -5 degrees) was found to be quite unusual, but it explains well the importance of a branched side chain at position 1 for channel formation and stability. A van der Waals interaction between valine-1 of one monomer and alanine-5 of the other helps to stabilize the gramicidin dimer.     

13C solid-state NMR of gramicidin A in a lipid membrane.

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

The structure of an integral membrane peptide: a deuterium NMR study of gramicidin.

Solid state deuterium NMR was employed on oriented multilamellar dispersions consisting of 1,2-dilauryl-sn-glycero-3-phosphatidylcholine and deuterium (2H) exchange-labeled gramicidin D, at a lipid to protein molar ratio (L/P) of 15:1, in order to study the dynamic structure of the channel conformation of gramicidin in a liquid crystalline phase. The corresponding spectra were used to discriminate between several structural models for the channel structure of gramicidin (based on the left- and right-handed beta 6.3 LD helix) and other models based on a structure obtained from high resolution NMR. The oriented spectrum is complicated by the fact that many of the doublets, corresponding to the 20 exchangeable sites, partially overlap. Furthermore, the asymmetry parameter, eta, of the electric field gradient tensor of the amide deuterons is large (approximately 0.2) and many of the amide groups are involved in hydrogen bonding, which is known to affect the quadrupole coupling constant. In order to account for these complications in simulating the spectra in the fast motional regime, an ab initio program called Gaussian 90 was employed, which permitted us to calculate, by quantum mechanical means, the complete electric field gradient tensor for each residue in gramicidin (using two structural models). Our results indicated that the left-handed helical models were inconsistent with our observed spectra, whereas a model based on the high-resolution structure derived by Arseniev and coworkers, but relaxed by a simple energy minimization procedure, was consistent with our observed spectra. The molecular order parameter was then estimated from the motional narrowing assuming the relaxed (right-handed) Arseniev structure. Our resultant order parameter of SZZ = 0.91 translates into an rms angle of 14 degrees, formed by the helix axis and the local bilayer normal. The strong resemblance between our spectra (and also those reported for gramicidin in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilayers) and the spectra of the same peptide incorporated in a lyotropic nematic phase, suggests that the lyotropic nematic phase simulates the local environment of the lipid bilayer.     

The pore dimensions of gramicidin A.

The ion channel forming peptide gramicidin A adopts a number of distinct conformations in different environments. We have developed a new method to analyze and display the pore dimensions of ion channels. The procedure is applied to two x-ray crystal structures of gramicidin that adopt distinct antiparallel double helical dimer conformations and a nuclear magnetic resonance (NMR) structure for the beta6.3 NH2-terminal to NH2-terminal dimer. The results are discussed with reference to ion conductance properties and dependence of pore dimensions on the environment.     

1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices

The structure of [Val1]gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer (Formula: see text) formed by two right-handed, single-stranded beta 6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.     

Role of Side-Chain Conformational Entropy in Transmembrane Helix Dimerization of Glycophorin A

Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of β-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix. Deuterium NMR spectroscopy is used to characterize the dynamics of fully deuterated Val80 and Val84, two essential amino acids of the dimerization motif. Deuterium spectra of the glycophorin A transmembrane dimer were obtained using synthetic peptides corresponding to the transmembrane sequence containing either perdeuterated Val80 or Val84. These data were compared with spectra of monomeric glycophorin A peptides deuterated at Val84. In all cases, the deuterium line shapes are characterized by fast methyl group rotation with virtually no motion about the C_α-C_β bond. This is consistent with restriction of the side chain in both the monomer and dimer due to intrahelical packing interactions involving the β-methyl groups, and indicates that there is no energy cost associated with dimerization due to loss of conformational entropy. In contrast, deuterium NMR spectra of Met81 and Val82, in the lipid interface, reflected greater motional averaging and fast exchange between different side-chain conformers.     

Designing of peptides with left handed helical structure by incorporating the unusual amino acids

The conformational behaviour of delta Ala has been investigated by quantum mechanical method PCILO in the model dipeptide Ac-delta Ala-NHMe and in the model tripeptides Ac-X-delta Ala-NHMe with X = Gly, Ala, Val, Leu, Abu and Phe and is found to be quite different. The computational results suggest that in the model tripeptides the most stable conformation corresponds to phi 1 = -30 degrees, psi 1 = 120 degrees and phi 2 = psi 2 = 30 degrees in which the > C = 0 of the acetyl group is involved in hydrogen bond formation with N-H of the amide group. Similar results were obtained for the conformational behaviour of D-Ala in Ac-D-Ala-NHMe and Ac-Ala-D-Ala-NHMe. The conformational behaviour of the amino acids delta Ala, D-Ala, Val and Aib in model tripeptides have been utilized in the designing of left handed helical peptides. It is shown that the peptide HCO-(Ala-D-Ala)3-NHMe can adopt both left and right handed helix whereas in the peptide Ac-(Ala-delta Ala)3-NHMe the lowest energy conformer is beta-bend ribbon structure. Left handed helical structure with phi = 30 degrees, psi = 60 degrees for D-Ala residues and phi = psi = 30 degrees for delta Ala is found to be more stable by 4 kcal mole-1 than the corresponding right handed helical structure for the peptide Ac-(D-Ala-delta Ala)3-NHMe. In both the peptides Ac-(Val-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe the most stable conformer is the left handed helix. Comparisons of results for Ac-(Ala-delta Ala)3-NHMe and Ac(Val-delta Ala)3-NHMe and Ac-(D-Ala-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe also reveal that the Val residues facilitate the population of 3(10) left handed helix over the other conformers. It is also shown that the conformational behaviour of Aib residue depends on the chirality of neighbouring amino acids, i.e. Ac-(Aib-Ala)3-NHMe adopts right handed helical structure whereas Ac-(Aib-D-Ala)3-NHMe is found to be in left handed helical structure.     

Determination of the structure of a membrane-incorporated ion channel. Solid-state nuclear magnetic resonance studies of gramicidin A.

Solid-state nuclear magnetic resonance (NMR) measurements on 13C-labeled analogues of the ion channel-forming peptide, gramicidin A, have been used to directly determine the structure of this peptide in lipid membranes. Seven gramicidin analogues, each labeled in a single carbonyl group of gly2, L-ala3, D-leu4, L-val7, D-leu10, D-leu12, or D-leu14 were synthesized by the solid-phase method. These gramicidin analogues were incorporated into aligned multilayers of dimyristoylphosphatidylcholine, or diether lipid bearing 14- or 16-carbon chains, at a 1:15 peptide:lipid mole ratio. Proton-enhanced, 13C, solid-state spectra were obtained at several temperatures and over a range of sample orientations with respect to the spectrometer magnetic field to permit accurate measurement of the chemical shift anisotropies. The observed anisotropies indicate that all of the labeled carbonyl bonds are oriented almost parallel to the molecular long axis and perpendicular to the lipid bilayer plane. These orientations are consistent with gramicidin forming a beta 6.3 single-strand helix that is oriented parallel to the methylene chains of the lipid molecules. Comparison of the linewidths from labeled residues that are in the innermost turn of the helix (gly2, ala3, and D-leu4), in the center of the molecule (val7), and in the turn nearest the lipid bilayer surface (D-leu10, D-leu12, and D-leu14) suggests that although the peptide behaves largely as a rigid barrel, segments of the peptide close to the membrane surface possess greater motional freedom.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta 6.3 channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi 1 and chi 2 angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi 1, chi 2) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi 2) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi 1, chi 2) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).     

Conformational study of peptides containing dehydrophenylalanine: helical structures without hydrogen bond

The conformational behaviour of deltaZPhe has been investigated in the model dipeptide Ac-deltaZPhe-NHMe and in the model tripeptides Ac-X-deltaZPhe-NHMe with X=Gly,Ala,Val,Leu,Abu,Aib and Phe and is found to be quite different. In the model tripeptides with X=Ala,Val,Leu,Abu,Phe the most stable structure corresponds to phi1=-30 degrees, psi1=120 degrees and phi2=psi2=30 degrees. This structure is stabilized by the hydrogen bond formation between C=O of acetyl group and the NH of the amide group, resulting in the formation of a 10-membered ring but not a 3(10) helical structure. In the peptides Ac-Aib-deltaZPhe-NHMe and Ac-(Aib-deltaZPhe)3-NHMe, the helical conformers with phi = +/-30 degrees, psi = +/-60 degrees for Aib residue and phi=psi= +/-30 degrees for deltaZPhe are predicted to be most stable. The computational studies for the positional preferences of deltaZPhe residue in the peptide containing one deltaZPhe and nine Ala residues reveal the formation of a 3(10) helical structure in all the cases with terminal preferences for deltaZPhe. The conformational behaviour of Ac-(deltaZPhe)n-NHMe with n< or =4 is predicted to be very labile. With n > 4, degenerate conformational states with phi,psi values of 0 degrees +/- 90 degrees adopt helical structures which are stabilized by carbonyl-carbonyl interactions and the N-H-pi interactions between the amino group of every deltaZPhe residue with one C-C edge of its own phenyl ring. The results are in agreement with the experimental finding that screw sense of helix for peptides containing deltaZPhe residues is ambiguous in solution. The helical structures stabilized by hydrogen bond formation are found to be at least 3kCalmol(-1) less stable. Conformational studies have also been carried out for the peptide Ac-(deltaEPhe)6-NHMe and the peptide Ac-deltaAla-(deltaZPhe)6-NHMe containing deltaAla residue at the N-terminal. The N-H-pi interactions are absent in peptide Ac-(deltaEPhe)6-NHMe.     

Induction of morphological changes in model lipid membranes and the mechanism of membrane disruption by a large scorpion-derived pore-forming peptide

The membrane disruption mechanism of pandinin 1 (pin1), an antimicrobial peptide isolated from the venom of the African scorpion, was studied using 31P, 13C, 1H solid-state and multidimensional solution-state NMR spectroscopy. A high-resolution NMR solution structure of pin1 showed that the two distinct alpha-helical regions move around the central hinge region, which contains Pro19. 31P NMR spectra of lipid membrane in the presence of pin1, at various temperatures, showed that pin1 induces various lipid phase behaviors depending on the acyl chain length and charge of phospholipids. Notably, it was found that pin1 induced formation of the cubic phase in shorter lipid membranes above Tm. Further, the 13C NMR spectra of pin1 labeled at Leu28 under magic angle spinning (MAS) indicated that the motion of pin1 bound to the lipid bilayer was very slow, with a correlation time of the order of 10(-3) s. 31P NMR spectra of dispersions of four saturated phosphatidyl-cholines in the presence of three types of pin1 derivatives, [W4A, W6A, W15A]-pin1, pin1(1-18), and pin1(20-44), at various temperatures demonstrated that all three pin1 derivatives have a reduced ability to trigger the cubic phase. 13C chemical shift values for pin1(1-18) labeled at Val3, Ala10, or Ala11 under static or slow MAS conditions indicate that pin1(1-18) rapidly rotates around the average helical axis, and the helical rods are inclined at approximately 30 degrees to the lipid long axis. 13C chemical shift values for pin1(20-44) labeled at Gly25, Leu28, or Ala31 under static conditions indicate that pin1(20-44) may be isotropically tumbling. 1H MAS chemical shift measurements suggest that pin1 is located at the membrane-water interface approximately parallel to the bilayer surface. Solid-state NMR results correlated well with the observed biological activity of pin1 in red blood cells and bacteria.     

Conformation of gramicidin in relation to its ability to form bilayers with lysophosphatidylcholine

The ability of gramicidin to induce bilayer formation in lysophosphatidylcholine (LPC) systems was investigated as a function of the conformation of the peptide. The conformation was varied by using different solvents to cosolubilize gramicidin and lipid. Using circular dichroism (CD), it was found that when codissolved in trifluoroethanol (TFE), after drying and subsequent hydration, gramicidin is mainly present in the single-stranded beta 6.3-helical configuration, whereas when using chloroform/methanol or ethanol as the solvent, it is proposed that the dominant conformation of gramicidin in the membrane is that of the double-stranded antiparallel dimer. Employing 31P NMR, the stoichiometry for bilayer formation was found to be 6 to 7 lipid molecules per gramicidin monomer, when samples were prepared from TFE, whereas a stoichiometry of 4 was found when chloroform/methanol or ethanol was the solvent. Upon heating the latter samples, a conversion was observed in the CD pattern toward that indicative of the beta 6.3-helical configuration. This change was accompanied by an increase in the extent of bilayer formation. Next, it was investigated whether the conformation of gramicidin and its ability to induce bilayer formation were dependent on the lipid acyl chain length. CD measurements of samples prepared from TFE indicated that gramicidin, independent of acyl chain length, was present in the beta 6.3-helical configuration but the intensity of the ellipticities at 218 nm increased with the length of the acyl chain. The extent of bilayer formation in these samples was found to be largely chain length independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deuterium NMR investigation of an amphotericin B derivative in mechanically aligned lipid bilayers

The methyl-d(3) amide derivative of the polyene antibiotic amphotericin B was synthesized, assayed for biological activity, incorporated into mechanically aligned bilayers of dipalmitoylphosphatidylcholine (DPPC), and examined by deuterium and phosphorus NMR. The amide derivative has a lesser, but qualitatively similar, biological activity relative to amphotericin B. Incorporation of the amide derivative and ergosterol into aligned DPPC bilayers resulted in a single, stable bilayer phase, as shown by phosphorus NMR of the DPPC headgroups. Deuterium NMR spectra revealed one major (2)H quadrupolar splitting and one major (2)H-(1)H dipolar splitting in the liquid-crystalline phase, consistent with a high degree of alignment and a single, averaged physical state for amphotericin B methyl-d(3) amide in the bilayer. Variations of the quadrupolar and dipolar splittings as a function of macroscopic sample orientation and temperature indicated that the amide derivative undergoes fast rotation about a motional axis that is parallel to the bilayer normal.     

Solid-state NMR spin diffusion for measurement of membrane-bound peptide structure: gramicidin A

A recently developed solid-state NMR method for measurement of depths in membrane systems is applied to gramicidin A, a membrane-bound peptide of known structure, to investigate the potential of this method. (15)N-detected, (1)H spin diffusion experiments demonstrate the resolution of the technique by measuring the 4-5 A depth differences between three (15)N-labeled backbone sites (Trp13, Val7, Gly2) in gramicidin A. We also show that (13)C-detected, (1)H spin diffusion experiments on unlabeled gramicidin A are sufficient to discriminate between the end-to-end dimer and double-helix structures of gramicidin A. Thus, spin diffusion solid-state NMR experiments can provide a simple approach, which does not require labeled samples, for testing structural models of membrane-bound peptides.     

Solution conformation of a tetradecapeptide stabilized by two di‐n‐propyl glycine residues

The solution conformation of a designed tetradecapeptide Boc‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐OMe (Dpg‐14) containing two di‐n‐propyl glycine (Dpg) residues has been investigated by ¹H NMR and circular dichroism in organic solvents. The peptide aggregates formed at a concentration of 3 mM in the apolar solvent CDCl₃ were broken by the addition of 12% v/v of the more polar solvent DMSO‐d₆. Successive N_iH N_i+1H NOEs observed over the entire length of the sequence in this solvent mixture together with the observation of several characteristic medium‐range NOEs support a major population of continuous helical conformations for Dpg‐14. Majority of the observed coupling constants ( ) also support ? values in the helical conformation. Circular dichroism spectra recorded in methanol and propan‐2‐ol give further support in favor of helical conformation for Dpg‐14 and the stability of the helix at higher temperature. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Conformation of the eosinophil chemotactic tetrapeptides and analogues in dimethyl sulfoxide. 1H n.m.r. studies

Proton nuclear magnetic resonance parameters are reported for DMSO-d6 solutions of the eosinophil chemotactic tetrapeptides, Val1-Gly2-Ser3-Glu4 and Ala1-Gly2-Ser3-Glu4, as well as three analogues of the Val1 tetrapeptide, D-Val1, Ala2 and Ala3. The synthesis of Val-(S)-[alpha-2 H1] Gly-Ala-Glu, in which the glycine has been stereospecifically deuterated in the H alpha 3 position, has allowed the assignment of the 1H resonances belonging to individual H alpha 2 and H alpha 3 glycine methylene protons. Simulation of the glycine ABX spin system yields two vicinal coupling constants which are consistent with a highly preferred conformation about the glycine HN-C alpha bond. The chemical shifts, coupling constants, temperature coefficients of amide proton chemical shifts and calculated side chain rotamer populations are reported for all peptides. The coupling constant analysis and temperature coefficients of amide proton chemical shifts together suggest that a type I beta-turn conformation is preferred by the Ala3 analogue. The 1H n.m.r. parameters of the other peptides suggest that these can also adopt a beta-turn conformation in DMSO. There are, however, considerable differences in the extent of conformational averaging undergone by the various peptides.     

Solution structure, stability, and nucleic acid binding of the hyperthermophile protein Sso10b2

The Sso10b (or Alba) family of proteins is a conserved group of archaeal and eukaryotic proteins which are thought to play a role in both chromatin organization and RNA metabolism. We describe here the solution structure and properties of Sso10b2 from Sulfolobus solfataricus. NMR data including residual dipolar couplings and (15)N relaxation data demonstrated that the protein adopts a beta(1)alpha(1)beta(2)alpha(2)beta(3)beta(4) topology with an IF-3-like fold. The protein dimerizes in solution at 30 degrees C via a hydrophobic surface defined by the C-terminal alpha(2)beta(3)beta(4) elements with a structure similar to one of the putative dimers indicated by previous crystal structures. DSC and circular dichroism data demonstrated an unusual two-state structural transition near the growth temperature which led to an increase in beta-sheet content without dissociation of the dimer. The cooperativity of the transition exceeded that of a dimer at pH 7, demonstrating the presence of higher order oligomers near the growth temperature at pH 7. Reverse titrations of Sso10b2 with nucleic acid showed that the protein binds single-stranded DNA (K(d) of 3 x 10(-)(7) M) with higher affinity than RNA (1.3 x 10(-)(6) M) or double-stranded DNA (1.5 x 10(-)(5) M) in 10 mM KH(2)PO(4) (pH 7.0, 20 degrees C). NMR chemical shift perturbation data indicated that single-stranded DNA and RNA binding occurred across the same dimer interface and encompassed a surface defined by the C-terminal ends of the beta(1), beta(2), and beta(3) strands of each monomer.     

Solution NMR structure of a model class A (apolipoprotein) amphipathic alpha helical peptide

To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH(2) (acetyl-AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d(3))/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for C(beta)H protons (all the residues except Ala and Val) and gammaCH(3) (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic alpha helical structure of Ac-18A-NH(2) is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix.