Biomedical subjects

B D Sykes

Publications and source records attributed to B D Sykes.

At least 19 recordsLinked to original sources

PubMed1997

Insight into lipid surface recognition and reversible conformational adaptations of an exchangeable apolipoprotein by multidimensional heteronuclear NMR techniques.

Apolipophorin III (apoLp-III) from the insect Manduca sexta is a 166-residue (Mr 18,340) member of the exchangeable apolipoprotein class that functions to stabilize lipid-enriched plasma lipoproteins. In the present study, we present the secondary structure and global fold of recombinant apoLp-III derived from three-dimensional heteronuclear NMR spectroscopy experiments. Five discrete alpha-helical segments (21-30 residues in length) with well defined boundaries were characterized by four NMR parameters: medium range nuclear Overhauser enhancement contacts between proton pairs, chemical shift index, coupling constants, and amide proton exchange rates. An antiparallel arrangement of helical segments has been obtained based on the long range interhelical nuclear Overhauser enhancement contacts. The NMR solution structure reveals a globular, up and down helix bundle organization similar to that of Locusta migratoria apoLp-III (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608). However, a short helix (comprised of 5 amino acids) has been identified in the region between helix 3 and helix 4. This helix is postulated to play a role in lipid surface recognition and/or initiation of binding. Our results also indicate the existence of buried polar and charged residues in the helix bundle, providing a structural basis for the relatively low stability of apoLp-III in its lipid-free state. It is suggested that the intrinsic low stability of lipid-free apoLp-III may be important in terms of its ability to undergo a reversible, lipid binding-induced, conformational change. This study underscores the striking resemblance in molecular architecture between insect apoLp-III and the N-terminal domain of human apolipoprotein E. The potential for application of NMR techniques to studies of the exchangeable apolipoproteins, possibly in their biologically active, lipid-associated state, has broad implications in terms of our understanding of the molecular basis of their physiological functions.

Amino Acid Sequence↗

PubMed1997

Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain.

The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca2+-bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction.

Alanine↗

PubMed1997

Interactions of structural C and regulatory N domains of troponin C with repeated sequence motifs in troponin I.

The actomyosin ATPase inhibitory protein troponin I (TnI) plays a central regulatory role in skeletal and cardiac muscle contraction and relaxation through its calcium-dependent interactions with troponin C (TnC) and actin. Previously we have demonstrated the utility of F29W and F105W mutants of TnC for measurement of binding affinities of inhibitory peptide TnI(96-116) to its regulatory N and structural C domains, both in isolation and in the intact TnC molecule [Pearlstone, J. R. & Smillie, L. B. (1995) Biochemistry 34, 6932-6940]. This approach is now extended to fragment TnI(96-148). Curve-fitting analyses of fluorescence changes induced in the intact TnC mutants and the isolated N and C domains by increasing [TnI(96-148)] have permitted the assignments of K(D) values (designated K(D,N) and K(D,C)) to the interaction of TnI(96-148) with the N and C domains, respectively, of intact TnC. Taken together with the previous data for TnI(96-116) binding, it can be concluded that, within TnI(96-148), residues 96-116 are primarily responsible for binding to C domain of intact TnC and residues 117-148 to its N domain. Inspection of the available mammalian and avian skeletal muscle TnI amino acid sequences reveals a previously unrecognized conserved motif repeated 3-fold, once in the inhibitory peptide region (approximately residues 101-114; designated alpha) and twice more in the region of residues approximately 121-132 (beta) and approximately 135-146 (gamma). The number and distribution of these motifs have important structural implications for the TnI x C complex. In the beta motif of cardiac TnI, as compared with skeletal, several changes in charged amino acids are suggested as candidates responsible for the greater sensitivity of cardiac Ca2+-regulated actomyosin to acidic pH as in ischemia.

Amino Acid Sequence↗

PubMed1997

Mechanism of direct coupling between binding and induced structural change in regulatory calcium binding proteins.

The structural transition in troponin C induced by the binding of two calcium ions involves an "opening" of the structure, an event that triggers skeletal muscle contraction. We have solved the solution structure of a mutant (E41A) of the regulatory domain of skeletal troponin C wherein one bidentate ligand to the calcium in site I is missing. This structure remains "closed" upon calcium binding, indicating that the linkage between calcium binding and the induced conformational change has been broken. This provides a snapshot of skeletal troponin C between the off and on state and thereby valuable insight into the mechanism of regulation within skeletal TnC. Although several factors contribute to the triggering mechanism, the opening of the troponin C structure is ultimately dependent on one amino acid, Glu41. Insights into the structure of cardiac troponin C can also be derived from this skeletal mutant.

Alanine↗

PubMed1997

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design.

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.

Amino Acid Sequence↗

PubMed1997

A molecular basis for different interactions of marine toxins with protein phosphatase-1. Molecular models for bound motuporin, microcystins, okadaic acid, and calyculin A.

The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful liver tumor promoters and potent inhibitors of the catalytic subunits of protein phosphatase-1 and -2A (PP-1c and PP-2Ac). In marked contrast to microcystins, which interact covalently with PP-1 and PP-2A, the nodularins do not bind covalently to PP-1 and PP-2A and may additionally possess unique carcinogenic properties. The conformation of microcystin-LR has been determined in solution and bound to PP-1c. We show here that the free NMR solution structures of two distinct microcystin structural congeners (microcystin-LR and -LL) are remarkably similar to the bound crystal structure of microcystin-LR. We have exploited this finding by using Metropolis Monte Carlo modeling to dock the solution structures of microcystin-LL and the marine toxin motuporin (nodularin-V) onto the crystal structure of PP-1c. Both of these toxins occupy a position similar to that of microcystin-LR when bound to PP-1c. However, although there are relatively minor differences in the structural orientation of microcystin-LL compared with microcystin-LR, there is a striking difference in the position of the N-methyldehydrobutyrine residue in motuporin relative to the comparable N-methyldehydroalanine residue in microcystin-LR. We propose that this difference in orientation provides a molecular explanation for why nodularins are incapable of forming a covalent linkage with PP-1c. Furthermore, the predicted position of N-methyldehydrobutyrine in motuporin is at the surface of the PP-1c-toxin complex, which may thus facilitate chemical interaction with a further macromolecule(s) possibly relating to its carcinogenic properties. PP-1c and PP-2Ac are also targets for other marine toxins such as okadaic acid and calyculin A. It was therefore of interest to use Metropolis Monte Carlo modeling to dock the known free crystal structures of okadaic acid and calyculin A to the crystal structure of PP-1c. These experiments predict that both okadaic acid and calyculin A are strikingly similar to microcystins and motuporin in their tertiary structure and relative PP-1c binding position.

Animals↗

PubMed1997

Neutrophil-activating peptide-2 and melanoma growth-stimulatory activity are functional as monomers for neutrophil activation.

Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH --> NCH3). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca2+ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50-100 microM. Similarly, MGSA showed a monomer-dimer equilibrium with a Kd of approximately 5 microM. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers.

Chemokine CXCL1↗

PubMed1997

Injury and protection in split-thickness skin after very rapid cooling and warming.

The ability of low glycerol concentrations and high cooling and warming rates to optimize the survival of frozen/thawed split-thickness porcine skin was investigated. 1H nuclear magnetic resonance spectroscopy was used to measure the diffusion kinetics of glycerol in skin at 4, 12, and 22 degrees C. Equilibrium concentrations were 44 to 69% of the external bathing medium. Rate constants for glycerol diffusion (D/l2) were calculated from the uptake data using a plane sheet model and a least squares method and were independent of external glycerol concentrations: D/l2 = 3.84 x 10(-4) 8-1 at 4 degrees C with an activation energy of 11.2 +/- 4.3 kcal/mol. Skin was cooled rapidly (-5100 degrees C/min) after different times of glycerol permeation at 4 or 22 degrees C, and survival was assessed after warming (+5400 degrees C/min) by an oxygen consumption assay. Recovery of aerobic activity increased in a concentration-dependent manner, and reached 100% after a 10-min exposure to 2 M glycerol at 4 degrees C or 3 min at 22 degrees C, for an uptake of 1.1 M glycerol. Light micrographs of freeze-substituted skin showed a glycerol-dependent decrease in the nucleation and growth of ice in the dermis and epidermis after rapid cooling. A 5-mm exposure to 2 M glycerol at 22 degrees C resulted in the elimination of all observable epidermal ice, except for extremely small ice crystals (< or = 0.5 micron diameter) in the intercellular spaces and in few nuclei, and complete preservation of the fibrous structure of dermal collagen bundles. This cryoprotective mechanism has the potential to offer complete protection of both dermal and viable epidermal targets of freeze/thaw injury and may be applicable to other thin, membranous tissues.

Animals↗

PubMed1997

Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.

The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.

Apolipoprotein E3↗

PubMed1996

NMR characterization of side chain flexibility and backbone structure in the type I antifreeze protein at near freezing temperatures.

The flexibility of the polar side chains in the alpha-helical Type I antifreeze protein (AFP) near the solution freezing temperature was investigated by two-dimensional nuclear magnetic resonance spectroscopy. These experiments were conducted to define the rotameric conformations of the proposed ice-binding groups, threonines and asparagines, in order to probe the molecular mechanism for ice binding. On the basis of the 3J alpha beta 2 NMR coupling constant values of 7.1, 8.5, 8.5, and 6.8 Hz for residues T2, T13, T24, and T35, respectively, it can be calculated that the regularly spaced ice-binding threonines sample many possible rotameric states prior to ice binding. The lack of a dominant side chain rotamer is further corroborated by nuclear Overhauser distance measurements for T13 and T24. N16 and N27, both with 3J alpha beta 2 and 3J alpha beta 3 coupling constants of 8.4 and 4.5 Hz, respectively, show a slight preference for the side chain conformation with a chi 1 of -60 degrees. These data suggest that prior to ice binding the threonine and asparagine side chains are free to rotate and that a unique preformed ice-binding structure in solution is not apparent. These observations do not support the rigid side chain model proposed recently by an X-ray study [Sicheri, F., & Yang, D. S. C. (1995) Nature 375, 427-431].

Amino Acid Sequence↗

PubMed1996

NMR study of the transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor complex. Visualization of human TGF-alpha binding determinants through nuclear Overhauser enhancement analysis.

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.

ErbB Receptors↗

PubMed1996

1H NMR evidence that Glu-38 interacts with the N-terminal functional domain in interleukin-8.

In order to assess the importance of the buried Glu-38 observed in the structure of interleukin-8, an analog in which Glu-38 was replaced with Ala (E38A analog) was investigated by 1H NMR spectroscopy and neutrophil activation. Detailed analysis of the NMR NOESY data showed that the solution structure of the E38A analog is essentially the same as that for the native protein. Also, the neutrophil elastase activity of the E38A analog was similar to that of the native protein. However, the Gln-8 and Cys-9 amide proton chemical shifts, which are significantly downfield-shifted in the native protein, exhibit more 'normal' values. This observation indicates that in the native protein, Glu-38 side-chain carboxylate interacts with Gln-8 and Cys-9 amide protons. Although the N-terminal residues are critical for function, this interaction is not essential for neutrophil activation.

Amino Acid Sequence↗

PubMed1996

Differential binding of desmopressin and vasopressin to neurophysin-II.

Desmopressin is a synthetic analog of the peptide hormone vasopressin in which the N-terminal alpha-amino group has been removed and L-arginine in position 8 has been replaced by D-arginine. Using 1H-NMR spectroscopy, we show that desmopressin binds to neurophysin-II, whereas deamino-vasopressin does not bind. Thus, the change in configuration at Arg8 causes a significant difference in the binding of these hormones to neurophysin-II. We have determined the structure of desmopressin bound to neurophysin-II using two-dimensional 1H nuclear magnetic resonance-transferred nuclear Overhauser effect techniques. A common binding motif for vasopressin and desmopressin is proposed that includes a positive charge group along with the hydrophobic surface formed by the side chains of Tyr2 and a beta-methylene provided by Phe-3. In vasopressin, the positive charge is provided by the N-terminal NH3+, whereas in desmopressin, the side chain of Arg-8 contributes the positive charge. The type II beta-turn found in residues Cys6-Pro7-D-Arg8-Gly9 of the bound structure of desmopressin folds the Arg8 side chain back toward the disulfide-bond loop, which allows the positive charged side chain of Arg8 to participate in binding. Such a type II beta-turn is not found in deamino-vasopressin in the presence of neurophysin-II.

Animals↗

PubMed1996

Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction.

BACKGROUND: Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS: The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS: In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

Animals↗

PubMed1996

Structural characterization of a monomeric chemokine: monocyte chemoattractant protein-3.

1H-NMR spectroscopy and analytical ultracentrifugation studies reveal that monocyte chemoattractant protein-3 (MCP-3) is a monomer. NMR solution structure shows that MCP-3 adopts an alphabeta fold similar to what is observed in structures of other known chemokines. However, MCP-3 is unique in that it does not show a propensity to form dimers. The closely related chemokines MCP-1 and MCP-2 show a monomer-dimer equilibrium in sedimentation equilibrium studies (approximately 0.2-2 mg/ml). As these proteins are present at nanomolar concentrations in vivo, the results suggest that they are monomeric at functional concentrations and that the monomer is the functionally significant form of MCP-1, MCP-2 and MCP-3.

Amino Acid Sequence↗

PubMed1996

Lactam bridge stabilization of alpha-helices: the role of hydrophobicity in controlling dimeric versus monomeric alpha-helices.

A series of lactam-bridged and linear 14 residue amphipathic alpha-helical peptides based on the sequence Ac-EXEALKKEXEALKK-amide were prepared in order to determine the effect of decreasing the hydrophobicity of the nonpolar face to helical content and stability. This was done by substituting position X by Ile, Val, and Ala. Lactam bridges spaced i to i + 4 were formed between the side chains of Glu3 and Lys7 and Glu10 and Lys14 while the linear noncyclized peptides could potentially form i to i + 4 salt bridges with the same residues. It was found that in all cases the lactam-bridged peptides were substantially more helical than the corresponding linear peptides as determined by CD spectroscopy. Moreover, the helical content approached 100% for the lactam-bridged peptides X = Ile and Ala and was greater than 80% for X = Val. For X = Ile and Val, this was partly due to the ability of the lactam bridges to enhance interchain interactions relative to the linear versions of the same sequence. Size-exclusion chromatography demonstrated that the Ile-based peptide associates as a dimer. The alanine-based lactam-bridged peptide was found to be monomeric as determined by concentration dependency studies and size-exclusion chromatography. Thermal denaturation studies in benign media indicated that the lactam-based peptides were very stable. The conformation of the Ala-based lactam peptide was further characterized by two-dimensional NMR spectroscopy and was found to be highly helical. The results demonstrate the ability of lactam bridges to stabilize the helical conformation and enhance dimerization of peptides based on a 3,4 hydrophobic heptad repeat. The substitution of Ala residues in the hydrophobic face of the alpha-helix can prevent dimerization and specify monomeric helical structure.

Amino Acid Sequence↗

PubMed1996

NMR solution structure of a synthetic troponin C heterodimeric domain.

The C-terminal domain from the muscle protein troponin C (TnC) comprises two helix-loop-helix calcium-binding sites (residues 90-162). The assembly of these two sites is governed by calcium binding enabling a synthetic C-terminal domain to be preferentially and stoichiometrically assembled from two synthetic peptides (residues 93-126, SCIII, and 129-162, SCIV) in the presence of calcium only. It is therefore of great interest to know how closely the structure of this heterodimeric domain is to the intact protein domain. Analysis of such a structure has important implications in protein engineering and in understanding the stability of calcium-binding proteins in terms of biological function. The solution structure of this heterodimeric protein was determined by 1H NMR spectroscopy using 802 NOE derived distance restraints and 23 phi and 22 chi angle restraints. Distance geometry-simulated annealing calculations yielded a family of 42 converged structures (rmsd 0.86 +/- 0.17 A) showing an arrangement of four alpha-helices similar in fold to the C-terminal of troponin C. The dimer interface has several important interactions between helix pairs E/H and F/G responsible for the association of the two peptides. However, neither the peptide complex nor the solution NMR structure of TnC pack as tightly as that observed in the TnC X-ray structure. The interhelical distance between the F/G helix is about 1.4 A greater in solution than in the crystal. A comparison of the exposed surface area of the hydrophobic residues in the SCIII/SCIV heterodimer revealed that residues 1104, Y112, and 1121 are more exposed than in the previously determined solution structure of the SCIII homodimer. These residues are important for the interaction with the inhibitory region of TnI and provide evidence for their involvement in the regulation of muscle contraction.

Amino Acid Sequence↗

PubMed1996

Assignment of the helical structure in neuropeptide Y by HPLC studies of methionine replacement analogues and 1H-NMR spectroscopy.

The HPLC retention behavior of three complete single methionine and methionine sulfoxide replacement sets of two 18-mer model peptides and neuropeptide Y (NPY) were investigated. All peptides were prepared by multiple solid-phase peptide synthesis. Plotting the retention time differences between methionine and methionine sulfoxide analogues vs the position of replacement shows that potentially alpha-helical peptides become helical on binding during reversed-phase high performance liquid chromatography. In the case of an amphipathic alpha-helix, the retention time differences change periodically with a 3-4 repeat pattern, which allow the location of amphipathic helical structures. Replacements in nonamphipathic alpha-helical domains cause local preferential binding areas and lead to sequence-dependent retention time profiles. Methionine replacement studies of NPY suggest an unstructured or extended conformation from Tyr1 to Ala12 connected to a well-defined amphipathic alpha-helix from Pro13 to Arg35. The assignment is confirmed by comparison of nuclear Overhauser effects based two-dimensional 1H-nmr spectroscopy and utilization of the C alpha H shift index method in 50% trifluoroethanol/50% water.

Amino Acid Sequence↗