1H NMR and CD secondary structure analysis of cell adhesion promoting peptide F-9 from laminin

A heparin binding, cell adhesion promoting domain, termed peptide F-9, from the B1 chain of human laminin, residues 641 to 660, i.e. RYVVLPRPVCFEKGMNYTVR, has been investigated by 1H NMR (500 MHz) spectroscopy and CD spectropolarimetry. While small linear peptides in water solution normally exist in a number of fluctuating conformational states, CD data analysis of peptide F9 indicates the existence of some preferred average structural populations consisting of about 30% beta-sheet, 22% beta-turn, and 6% alpha-helix. NMR structural analysis supports this observation and indicates specific sequences of preferred structural populations. Evidence for these is indicated by the presence of dNN nuclear Overhauser effect (NOE) populations and attenuated or absent d alpha N NOEs at short mixing times (0.1 s), 3J alpha N coupling constants of 5 and 10 Hz, and chemical shifts significantly removed from random coil positions. The NH2-terminal VVL sequence primarily exists in an extended chain conformation by virtue of large d alpha N NOEs and 9-10 Hz 3J alpha N coupling constants. Residues C10-N16 have turn-like or helix character with a run of dNN and d beta N NOEs and attenuated d alpha N NOEs. These midchain reversals include the lysine and asparagine residues proposed to be involved in heparin binding and N-glycosylation, respectively, to laminin peptide F-9.     

1H-NMR assignment and secondary structure of human insulin-like growth factor-I (IGF-I) in solution.

Human insulin-like growth factor-I (IGF-I) was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for all the backbone protons and almost all of the sidechain protons of the total 70 amino acid residues, using sequence-specific assignment procedures. The secondary structure elements of human IGF-I were identified by investigation of the sequential and medium range NOEs as a preliminary step in determining the three-dimensional structure of this protein by means of distance geometry calculations. The typical NOEs of d alpha beta(i,i + 3) and d alpha N(i,i + 3), as well as the successive strong NOEs of dNN connectivities and slowly exchanging amide protons confirmed the presence of three helical segments corresponding to the sequence regions, Ala8-Cys18, Gly42-Cys48, and Leu54-Cys61, and the existence of a beta-turn in the Gly19-Gly22 region. Our results definitely indicate that the secondary structure of human IGF-I in solution is consistent with that of insulin in the crystalline state.     

Two-dimensional NMR, circular dichroism, and fluorescence studies of PP-50, a synthetic ATP-binding peptide from the beta-subunit of mitochondrial ATP synthase.

PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD = 17.5 microM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong d alpha N NOEs, J alpha N values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i,i+2), and d alpha N(i,i+2) NOEs and 3J alpha N values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol.

A 22-residue synthetic peptide encompassing the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.     

Conformational features of a hexapeptide model Ac-TGAAKA-NH2 corresponding to a hydrated alpha helical segment from glyceraldehyde 3-phosphate dehydrogenase: implications for the role of turns in helix folding

Recent analysis of alpha helices in protein crystal structures, available in literature, revealed hydrated alpha helical segments in which, water molecule breaks open helix 5-->1 hydrogen bond by inserting itself, hydrogen bonds to both C=O and NH groups of helix hydrogen bond without disrupting the helix hydrogen bond, and hydrogen bonds to either C=O or NH of helix hydrogen bond. These hydrated segments display a variety of turn conformations and are thought to be 'folding intermediates' trapped during folding-unfolding of alpha helices. A role for reverse turns is implicated in the folding of alpha helices. We considered a hexapeptide model Ac-1TGAAKA6-NH2 from glyceraldehyde 3-phosphate dehydrogenase, corresponding to a hydrated helical segment to assess its role in helix folding. The sequence is a site for two 'folding intermediates'. The conformational features of the model peptide have been investigated by 1H 2D NMR techniques and quantum mechanical perturbative configuration interaction over localized orbitals (PCILO) method. Theoretical modeling largely correlates with experimental observations. Based upon the amide proton temperature coefficients, the observed d alpha n(i, i + 1), d alpha n(i, i + 2), dnn(i, i + 1), d beta n(i, i + 1) NOEs and the results from theoretical modeling, we conclude that the residues of the peptide sample alpha helical and neck regions of the Ramachandran phi, psi map with reduced conformational entropy and there is a potential for turn conformations at N and C terminal ends of the peptide. The role of reduced conformational entropy and turn potential in helix formation have been discussed. We conclude that the peptide sequence can serve as a 'folding intermediate' in the helix folding of glyceraldehyde 3-phosphate dehydrogenase.     

Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-1 envelope glycoprotein gp120

With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

A helix-turn motif in the C-terminal domain of histone H1

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.     

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Plastocyanin is a predominantly beta-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,Halpha) coupling constants, (15)N-edited proton-proton nuclear Overhauser effects (NOEs), and (15)N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to beta-strands in the folded protein exhibit small upfield shifts of the (13)C(alpha) and (13)CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i + 1) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the (13)C(alpha), (13)CO, and (1)H(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,Halpha) coupling constants. The (15)N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form beta-strands in the native protein spontaneously populate the beta-region of (phi,psi) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.     

Solution structure and backbone dynamics of long-[Arg(3)]insulin-like growth factor-I

Long-[Arg(3)]insulin-like growth factor-I (IGF-I) is a potent analog of insulin-like growth factor-I that has been modified by a Glu(3) --> Arg mutation and a 13-amino acid extension appended to the N terminus. We have determined the solution structure of (15)N-labeled Long-[Arg(3)]-IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 A root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 A root mean square deviation for all backbone heavy atoms excluding the 8 N-terminal residues and the 8 C-terminal eight residues. Overall, the structure of the IGF-I domain is consistent with earlier studies of IGF-I with some minor changes remote from the N terminus. The major variations in the structure, compared with IGF-I, occur at the N terminus with a substantial reorientation of the N-terminal three residues of the IGF-I domain. These results are interpreted in terms of the lower binding affinity for insulin-like growth factor-binding proteins. The backbone dynamics of Long-[Arg(3)]IGF-I were investigated using (15)N nuclear spin relaxation and the heteronuclear nuclear Overhauser enhancement (NOE). There is a considerable degree of flexibility in Long-[Arg(3)]IGF-I, even in the alpha-helices, as indicated by an average ((1)H)(15)N NOE of 0.55 for the regions. The largest heteronuclear NOEs are observed in the helical regions, lower heteronuclear NOEs are observed in the C-domain loop separating helix 1 from helix 2, and negative heteronuclear NOEs are observed in the N-terminal extension and at the C terminus. Despite these data indicating conformational flexibility for the N-terminal extension, slow amide proton exchange was observed for some residues in this region, suggesting some transitory structure does exist, possibly a molten helix. A certain degree of flexibility may be necessary in all insulin-like growth factors to enable association with various receptors and binding proteins.     

Sequence-dependent conformations of DNA duplexes: the TATA segment of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

The base and sugar protons of the d(G-G-T-A-T-A-C-C) duplex have been assigned from two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements in D₂O solution at 25°C. The nucleic acid protons have been assigned from NOEs between protons on adjacent bases on the same and partner strands, as well as from NOEs between the base protons and their own and 5′-flanking H1′, H2′, H2″, H3′, and H4′ sugar protons. These assignments are confirmed from coupling constant and NOE connectivities within the sugar protons of a given residue. Several of these NOEs exhibit directionality and demonstrate that the d(G-G-T-A-T-A-C-C) duplex is a right-handed helix. The relative magnitude of the NOEs between the base protons and the sugar H2′ protons of its own and 5′-flanking sugar demonstrate that the TATA segment of the d(G-G-T-A-T-A-C-C) duplex adopts a B-DNA type helix geometry in solution, in contrast to the previous observation of a A-type helix for the same octanucleotide duplex in the crystalline state.     

Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.     

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.     

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.     

Conformational stability of a thrombin-binding peptide derived from the hirudin C-terminus.

The COOH-terminal region of hirudin represents an independent functional domain that binds to an anion-binding exosite of thrombin and inhibits the interaction of thrombin with fibrinogen and regulatory proteins in blood coagulation. The thrombin-bound structure of the peptide fragment, hirudin 55-65, has been determined by use of transferred NOE spectroscopy [Ni, F., Konishi, Y., & Scheraga, H. A. (1990) Biochemistry 29, 4479-4489]. The stability of the thrombin-bound conformation has been characterized further by a combined NMR and theoretical analysis of the conformational ensemble accessible by the hirudin peptide. Medium- and long-range NOE's were found for the free hirudin peptide in aqueous solution and in a mixture of dimethyl sulfoxide and water at both ambient (25 degrees C) and low (0 degrees C) temperatures, suggesting that ordered conformations are highly populated in solution. The global folding of these conformations is similar to that in the thrombin-bound state, as indicated by NOE's involving the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Residues Glu(61), Glu(62), Tyr(63), and Leu(64) all contain approximately 50% of helical conformations calculated from the ratio of the sequential dNN and d alpha N NOE's. Among the helical ensemble, active 3(10)-helical conformations were found by an analysis of the medium-range [(i,i+2) and (i,i+3)] NOE's involving the last six residues of the peptide. An analysis of the side-chain rotamers revealed that, upon binding to thrombin, there may be a rotation around the alpha CH-beta CH bond of Ile(59) such that Ile(59) adopts a gauche- (chi 1 = +60) conformation in contrast to the highly populated trans (chi 1 = -60) found for Ile(59) in the free peptide. However, the thrombin-bound conformation of the hirudin peptide is still an intrinsically stable conformer, and the preferred conformational ensemble of the peptide contains a large population of the active conformation. The apparent preference for a gauche- (chi 1 = +60) side-chain conformation of Ile(59) in the bound state may be explained by the existence of a positively charged arginine residue among the hydrophobic residues in the thrombin exosite.     

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.     

Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix

The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain protons were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolve many ambiguities in intraresidue J-coupled connectivities and interresidue NOE connectivities. This required no explicit knowledge of the solution structure. The pattern of NOEs found is consistent with a regular alpha helix between glycine-6 and lysine-13; H bonding continues at least through alanine-15 [see Wand, A.J., Roder, H., & Englander, S. W. (1986) Biochemistry (following paper in this issue)]. Chain disorder occurs at the N-terminus. There is no indication of significant spin diffusion among the backbone amide and alpha-protons of this 12.4-kilodalton protein even at the longest NOE mixing time used (140 ms).     

Nuclear Overhauser effects as probes of peptide structure. A diagnostic for left-handed helical conformations

The conformational dependence of the interresidue interproton distances in peptides, C alpha H ... Ni + 1 H and NiH ... Ni + 1 H, have been used to identify zones of sterically allowed phi, psi space, where both distances are less than 3A and expected to yield nuclear Overhauser effects (NOEs). L-residues in left-handed helical conformations are expected to yield both interresidue NOEs and also an appreciable intraresidue NiH----C alpha iH NOE. The effect of cutoff distances has been evaluated. Experimental results on three model peptides illustrate the utility of these NOEs in identifying L-residues at the i + 2 position of Type II and I' beta-turns. Simultaneous observation of both interresidue NOEs may also be indicative of conformational heterogeneity in specific cases, as illustrated for a single residue in a decapeptide.     

NMR studies of model peptides of PHGGGWGQ repeats within the N-terminus of prion proteins: a loop conformation with histidine and tryptophan in close proximity

The N-terminal region of the prion protein from human and mouse contains five tandem repeats with the consensus sequence of PHGGGWGQ. NMR studies were performed in water for two cyclic peptides, cyclo-[C(1)R(2)Q(3)P(4)H(5)G(6)G(7)S(8)W(9)G(10)Q(11)R(12)D(13)C(14)] (C1) and cyclo-[C(1)R(2)D(3)P(4)H(5)G(6)G(7)G(8)W(9)G(10)Q(11)P(12)H(13)G(14)G (15)G(16)W(17)G(18)Q(19)R(20)D(21)C(22)] (C2), which are cyclized by a disulfide bridge between the Cys residues at the N- and C-termini, and for their corresponding linear peptides (L1 and L2) which are formed by reduction. The patterns of the C(alpha)H chemical shift difference of these four peptide mimetics were very similar to those observed for the tandem repeats of human prion protein reported by other researchers. The medium-range NOE connectivities were found between the C(beta)H of the H5 and the proton of the W9 side chain for L1. The corresponding NOEs were also observed in H5-W9 and H13-W17 of L2 with ambiguity. These observations indicate that histidine (i) is in close proximity to tryptophan (i+4). d(alphaN) (i,i+2) NOE connectivities were observed between W9 and Q11 of L1 and L2, and d(NN) (i,i+1) NOE connectivities were also observed for G10-Q11 of L1 and L2 and for G18-Q19 of L2. Significantly lower temperature coefficients of amide proton chemical shifts were obtained for Q11 and Q19 of L2 and C2. Structure calculations for L1 showed that HGG(G/S)W and (G/S)WGQ adopt a loop conformation and a beta-turn, respectively. These results strongly suggest that the tandem repeats within prion protein adopt a non-random structure.     

NMR structure and dynamics of the second transmembrane domain of the neuronal acetylcholine receptor beta 2 subunit

Structure and backbone dynamics of a selectively [(15)N]Leu-labeled 28-residue segment of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit were studied by (1)H and (15)N solution-state NMR in dodecylphosphocholine micelles. The TM2e structure was determined on the basis of the nuclear Overhauser effects (NOEs) and the hydrogen bond restraints, which were inferred from the presence of H(alpha)(i)-H(N)(i+3), H(alpha)(i)-H(beta)(i+3), and H(alpha)(i)-H(N)(i+4) NOE connectivity and from the slow amide hydrogen exchange with D(2)O. The TM2e structure of the nAChR beta(2) subunit contains a helical region between T4 and K22. Backbone dynamics were calculated using the model-free approach based on the (15)N relaxation rate constants, R(1) and R(2), and on the (15)N-[(1)H] NOE. The data acquired at 9.4 and 14.1 T and calculations using different dynamic models demonstrated no conformational exchange and internal motions on the nanosecond time scale. The global tumbling time of TM2e in micelles was 14.4 +/- 0.2 ns; the NOE values were greater than 0.63 at 9.4 T, and the order parameter, S(2), was 0.83-0.96 for all (15)N-labeled leucine residues, suggesting a restricted internal motion. This is the first report of NMR structure and backbone dynamics of the second transmembrane domain of the human nAChR beta(2) subunit in a membrane-mimetic environment, providing the basis for subsequent studies of subunit interactions in the transmembrane domain complex of the neuronal nAChR.     

Cystic fibrosis transmembrane conductance regulator: solution structures of peptides based on the Phe508 region, the most common site of disease-causing DeltaF508 mutation.

Most cases of cystic fibrosis (CF), a common inherited disease of epithelial cell origin, are caused by the deletion of Phe508 located in the first nucleotide-binding domain (NBF1) of the protein called CFTR (cystic fibrosis transmembrane conductance regulator). To gain greater insight into the structure within the Phe508 region of the wild-type protein and the change in structure that occurs when this residue is deleted, we conducted nuclear magnetic resonance (NMR) studies on representative synthetic 26 and 25 amino acid peptide segments. 2D 1H NMR studies at 600 MHz of the 26-residue peptide consisting of Met498 to Ala523 in 10% DMSO, pH 4.0, at 25 degrees C show a continuous but labile helix from Gly500 to Lys522, based on both NH-NH(i,i+1) and alphaH-NH(i,i+1) NOEs. Phe508 within this helix shows only short-range (i, 相似文献