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B D Sykes

Publications and source records attributed to B D Sykes.

At least 145 records · Page 8Linked to original sources
PubMed1993

Improved synthetic methods for the selective deuteration of aromatic amino acids: applications of selective protonation towards the identification of protein folding intermediates through nuclear magnetic resonance.

In this report we describe several novel methods for the preparation of selectively deuterated aromatic amino acids. New syntheses for [2,3,5,6-2H4]phenylalanine and [2,4,6,7-2H4]tryptophan, as well as improved catalytic exchange methods for [2,3,5,6-2H4]tyrosine and [2,3,4,5,6-2H5]phenylalanine are presented. Isotopic substitution levels for all compounds are generally found to be greater than 95%. Biosynthetic incorporation of these amino acids is also shown to be possible with little or no evidence of isotopic scrambling. The products from these new syntheses, in combination with other selectively deuterated aromatic amino acids, are found to permit group-specific 'single-proton' labelling of proteins. This highly-efficient and very cost-effective method of selective protonation is shown to produce greatly simplified 1H-NMR spectra of the aromatic region of proteins. The utility of this approach to isotopic editing is demonstrated with the identification of a transient folding intermediate of Escherichia coli thioredoxin which is undetectable by standard 2-D NMR techniques.

Deuterium↗
PubMed1993

A single-stranded amphipathic alpha-helix in aqueous solution: design, structural characterization, and its application for determining alpha-helical propensities of amino acids.

In order to investigate the positional effect of alpha-helical propensities of amino acids in an amphipathic alpha-helix, an amphipathic alpha-helical model peptide (Ac-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys- amide) was designed and characterized by circular dichroism and 2D-NMR spectroscopies. This peptide contains 65% alpha-helical structure in solution, and its monomeric molecular weight in aqueous solution was determined by size-exclusion chromatography. The independence of alpha-helical structure and stability on peptide concentration demonstrates that helix formation of this peptide is a monomolecular process. To compare the effect of substitutions in the hydrophobic and hydrophilic face of the helix on structure and stability, a leucine, alanine, or glycine was individually substituted in the hydrophobic face (position 9) or hydrophilic face (position 7) of the model peptide. The change in helix content and stability upon substitution was measured by circular dichroism studies in the absence and presence of TFE or urea. The results indicate that each amino acid has a different helix propensity when it is located in the hydrophobic face versus hydrophilic face and the effect of substitution is more significant in the hydrophobic face. This single-stranded amphipathic alpha-helical peptide provides an appropriate model system to determine helix propensities of amino acids on both hydrophobic and hydrophilic faces.

Amino Acid Sequence↗
PubMed1993

1H-NMR resonance assignments, secondary structure, and global fold of the TR1C fragment of turkey skeletal troponin C in the calcium-free state.

The TR1C fragment of turkey skeletal muscle TnC (residues 12-87) comprises the two regulatory calcium binding sites of the protein. Complete assignments of the 1H-NMR resonances of the backbone and amino acid side chains of this domain in the absence of metal ions have been obtained using 2D 1H-NMR techniques. Sequential (i,i+1) and short-range (i,i+3) NOE connectivities define two helix-loop-helix calcium binding motifs, and long-range NOE connectivities indicate a short two-stranded beta-sheet formed between the two calcium binding loops. The two calcium binding sites are different in secondary structure. In terms of helix length, site II conforms to a standard "EF-hand" motif with the first helix ending one residue before the first calcium ligand and the second helix starting one residue after the beta-sheet. In site I, the first helix ends three residues before the first calcium ligand, and the second helix starts three residues after the beta-sheet. A number of long-range NOE connectivities between the helices define their relative orientation and indicate formation of a hydrophobic core between helices A, B, and D. The secondary structure and global fold of the TR1C fragment in solution in the calcium-free state are therefore very similar to those of the corresponding region in the crystal structure of turkey skeletal TnC [Herzberg, O., & James, M.N.G. (1988) J. Mol. Biol. 203, 761-779].

Amino Acid Sequence↗
PubMed1993

Formation of the stable myosin-ADP-aluminum fluoride and myosin-ADP-beryllium fluoride complexes and their analysis using 19F NMR.

The effects of aluminum fluoride and beryllium fluoride on smooth muscle myosin and its subfragments were studied. Mg(2+)-ATPase activity was inhibited in the presence of aluminum fluoride (beryllium fluoride). [3H]ADP bound to heavy meromyosin (HMM) in the presence of aluminum fluoride (beryllium fluoride) and was not dissociated after 3 days of dialysis demonstrating that [3H]ADP was trapped in HMM. These results suggest the formation of a stable HMM-ADP-fluoroaluminate (fluoroberyllate) complex. The intrinsic tryptophane fluorescence intensity was increased in the presence of ADP and aluminum fluoride (beryllium fluoride). Acto-S1 was dissociated upon the formation of S1-ADP-fluoroberyllate and actin destabilized S1-ADP-fluoroberyllate complex, while S1-ADP-fluoroaluminate failed to bind to actin. Furthermore, when S1 formed the complex with actin, nucleotide trapping did not occur in the presence of fluoraluminate. These results indicated that the myosin-ADP-fluoroberyllate complex resembles a weak binding state while myosin-ADP-fluoroaluminate complex is a distinct conformation although the binding to actin was also weak. The structure of the ternary complex was investigated using 19F NMR. The 19F NMR spectrum of the S1-ADP-fluoroaluminate complex showed a peak at -66.7 ppm which is due to the binding of fluoraluminate to S1. The peak was not observed when 5'-adenylylimidodiphosphate was substituted for ADP suggesting that aluminum fluoride plays a role as a phosphate analogue. The stoichiometry of the bound fluoride was determined to be 3.8 mol/mol S1 suggesting that the bound species is AlF-4.

Adenosine Diphosphate↗
PubMed1993

The nonhelical structure of antifreeze protein type III.

Antifreeze proteins (AFPs) are present in the blood of some marine fishes and inhibit the growth of ice crystals at subzero temperatures by adsorption to the ice lattice. The solution structure of a Type III AFP was determined by two-dimensional nuclear magnetic resonance spectroscopy. These measurements indicate that this 66-residue protein has an unusual fold in which eight beta strands form two sheets of three antiparallel strands and one sheet of two antiparallel strands, and the triple-stranded sheets are packed orthogonally into a beta sandwich. This structure is completely different from the amphipathic, helical structure observed for Type I AFPs.

Amino Acid Sequence↗
PubMed1993

Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.

Amino Acid Sequence↗
PubMed1993

Determination of the conformations of bound peptides using NMR-transferred nOe techniques.

NMR-transferred nOe spectroscopy provides a means for determining the structure of a peptide bound to a macromolecular target when the complex is too large to be studied by conventional NMR techniques. Recent examples of structures that have been determined by NMR-transferred nOe techniques include platelet-receptor peptide bound to thrombin, cholera toxin peptide bound to antibodies, troponin I peptide bound to troponin C and the vsv-C peptide bound to chaperones DnaK and GroEL.

Amino Acid Sequence↗
PubMed1993

Characterization of gamma-radiation induced decomposition products of thymidine-containing dinucleoside monophosphates by nuclear magnetic resonance spectroscopy.

To study the chemical and biochemical influence of loss of base aromaticity, dinucleoside monophosphates containing cis-5R,6S-thymidine glycol (Tg) and 5R and 5S 5,6-dihydrothymidine (Th) were prepared from d-ApT and d-TpA by KMnO4 oxidation and rhodium-catalysed hydrogenation, respectively. One and two dimensional 1H NMR techniques were used to characterize the solution conformation of each of the modified dinucleoside monophosphates for comparison with the unmodified compounds. Coupling constant data show that all sugar moieties adopt a predominantly 2'-endo conformation. Estimates of proton-proton distances from two-dimensional NOE experiments reveal that most of the glycosidic bonds prefer the anti conformation. Analysis of the C5'-C4' (gamma) torsion angle of the hydroxymethyl group using 3JH4'H5' and 3JH4'H5" data indicate that these modifications to thymine have little effect on the gamma conformer populations. Although, in general, additions at C5 and C6 of thymine in d-ApT and d-TpA profoundly distort the pyrimidine, they do not otherwise significantly alter the conformation of these compounds relative to the unsubstituted dinucleoside monophosphates. The one exception is the thymine glycol of d-TgpA, which appears to have a higher syn population than the parent compound.

Carbohydrate Conformation↗
PubMed1992

Interaction of troponin I and troponin C: use of the two-dimensional transferred nuclear Overhauser effect to determine the structure of a Gly-110 inhibitory troponin I peptide analog when bound to cardiac troponin C.

The structure of a peptide analog of the inhibitory region of cardiac troponin-I (N-acetyl-G110-TnI(104-115) amide) when bound to cardiac troponin-C has been determined by 2-dimensional 1H-NMR techniques. The bound structure determined for this peptide is similar to that determined previously for the skeletal peptide (which has a proline at position 110) bound to skeletal troponin-C (Campbell and Sykes (1991) J. Mol. Biol. 222, 405-421). This structure shows a helical like peptide backbone 'bent' around P109-G110 to bring the hydrophobic residues F106, L111 and V114 closer together. The other 'side' of this structure is surrounded by the basic residues extending outwards towards the protein or solution. While the bound structures of the cardiac and skeletal peptides are shown to be quite similar, the cardiac peptide appears more flexible near the central glycine residue.

Amino Acid Sequence↗
PubMed1992

Determination of the solution structure of a synthetic two-site calcium-binding homodimeric protein domain by NMR spectroscopy.

The solution structure of a 34-residue synthetic calcium-binding peptide from site III of chicken troponin-C has been determined by 1H NMR spectroscopy. In solution and in the presence of calcium this peptide forms a symmetric two-site homodimeric calcium-binding domain (Shaw et al., 1990). The solution structure of this dimer was determined from the measurement of 470 NOEs from a 75-ms NOESY data set. For the dimer structure determination, the constraint list included 868 distance restraints, 44 phi angles, and 24 chi 1 and 2 chi 2 angles. Seven structures were calculated by restrained molecular dynamics using a procedure in which intramonomer distances were used first and then all distances, intra- and intermonomer, were input during further dynamics. The structures exhibited a fold very similar to the C-terminal domain of troponin-C comprised of a pair of helix-loop-helix calcium-binding sites. The rms deviation of these structures for backbone atoms between residues 97-122 and 97'-122' for the dimer was 0.82 A. The dimer structure was also calculated to be more symmetric than sites III and IV in troponin-C.

Amino Acid Sequence↗
PubMed1992

Spectroscopic analysis of a methionine-48 to tyrosine mutant of chicken troponin C.

A mutant (M48Y) of chicken skeletal muscle troponin C was prepared in which Tyr replaced Met-48 of the recombinant protein (rTnC). Since Tyr and Trp are normally absent, spectral properties could be unambiguously assigned to the site of substitution. In the crystal structure, this residue lies at the COOH-terminal end of the B-helix of the N domain in a region postulated to undergo a significant conformational change to a more polar environment upon Ca2+ binding [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644]. Comparison of the far-UV CD spectra of M48Y and rTnC in the absence and presence of Ca2+ indicated no overall structural alteration due to the mutation. However, Ca2+ titration of the ellipticity change showed a reduction in Ca2+ affinity and cooperativity of sites I and II. A Ca(2+)-induced increase in the near-UV ellipticity of M48Y at pH 7.12 and a red shift in its UV absorbance spectrum occurred over a range of free [Ca2+] attributable to the N-domain transition only. This was largely abolished at pH 5.3 where Ca2+ no longer binds to sites I and II. That region of the 1H NMR spectrum attributable to Tyr was broadened upon Ca2+ binding. These Ca(2+)-induced changes are consistent with the environment of the Tyr side chain becoming chiral, less polar, and more immobile, all in a direction opposite to that predicted. These observations indicate that while the general features of the postulated model are valid, it is unlikely to be correct in detail.

Animals↗
PubMed1992

31P-NMR study of the phospholipid moiety of lipophorin subspecies.

31P-NMR spectra of four distinct subspecies of Manduca sexta hemolymph lipophorin revealed the presence of two resonances separated by 0.6 ppm. Phospholipid analysis of the lipoproteins showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present and their mass ratio correlated well to the intensity of the two resonances in each of the different subspecies. The two resonances persisted in 31P-NMR spectra of organic solvent extracts of lipophorin. These results, together with the fact that PE, but not PC, can form an intramolecular hydrogen bond between the phosphate oxygen and the amino group of ethanolamine, resulting in deshielding of the phosphorus nucleus (and a 0.6 ppm downfield shift), strongly suggest the resonances observed represent the PC and PE components of these lipoproteins. 31P-NMR line-width data obtained as a function of temperature and solvent viscosity were used to calculate the chemical shift anisotropy (delta sigma), intrinsic viscosity (eta'), and lateral diffusion coefficients (DT) of PC and PE in different lipophorin subspecies. eta' and DT for PC and PE were similar among high-density lipophorins but differed in low-density lipophorin (LDLp). These differences may be related to the large increase in diacylglycerol content in this particle and/or the association of up to 16 molecules of apolipophorin III. On the basis of the known lipid compositional differences between LDLp and high-density lipophorin subspecies, we propose that uptake of large amounts of diacylglycerol during LDLp formation results in partitioning of this lipid to the surface monolayer where it intercalates between phospholipid molecules. Diacylglycerol intercalation creates gaps between phospholipid head groups that expose the hydrophobic surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide.

The structure of a synthetic peptide comprising the 28 amino-terminal residues of actin has been examined by 1H-NMR and CD spectroscopy. The peptide is largely unstructured and flexible in solution but becomes increasingly structured at higher trifluoroethanol (TFE) concentrations. As judged by CD with the use of two additional peptides (actin 1-20 and actin 18-28), TFE induces formation of up to 48% helical content within residues 1-20, while residues 21-28 exhibit no helical propensity. Similar results were obtained by using NMR-derived distance information in restrained molecular dynamics calculations. The calculated structure of actin 1-28 peptide in 80% TFE is well defined for the first 23 residues with a backbone root mean square deviation of 0.5 A. Two helices are formed from residues 4-13 and 16-20, and a beta-turn is formed from residues 13-16. The N-terminal residues 1-3 exhibit increased flexibility and a helix-like conformation while the C-terminal residues 21-28 show no regular secondary structure. These results are compared with the predicted secondary structure and the structure of the corresponding sequence in the crystal structure of actin [Kabsch et al. (1990) Nature 347, 37-44]. The significance of the TFE-induced peptide structure is discussed.

Actins↗
PubMed1992

Assignment of amide 1H and 15N NMR resonances in detergent-solubilized M13 coat protein: a model for the coat protein dimer.

The major coat protein of the filamentous coliphage M13 is a 50-residue integral membrane protein. Detergent-solubilized M13 coat protein is a promising candidate for structure determination by nuclear magnetic resonance methods as the protein can be prepared in large quantities and the protein-containing micelle is reasonably small. Under the conditions of our experiments, SDS-bound coat protein exists as a dimer with an apparent molecular weight of 27,000. Broad lines and poor resolution in the 1H spectrum have led us to adopt an 15N-directed approach, in which the coat protein was labeled both uniformly with 15N and selectively with [alpha-15N]alanine, -glycine, -valine, -leucine, -isoleucine, phenylalanine, -lysine, -tyrosine, and -methionine. Nitrogen resonances were assigned as far as possible using carboxypeptidase digestion, double-labeling, and an independent knowledge of the amide proton exchange rates determined from neighboring assigned 13C-labeled carbonyl carbons. 1H/15N heteronuclear multiple quantum coherence (HMQC) spectroscopy of both uniform and site-selectively-labeled proteins subsequently correlated amide nitrogen with amide proton chemical shifts, and the assignments were completed sequentially from homonuclear NOESY and HMQC-NOESY spectra. The most slowly exchanging amide protons were shown to occur in a continuous stretch extending from methionine-28 to phenylalanine-42. This sequence includes most of the resonances of the hydrophobic core, although it is shifted toward the C-terminal end of the protein. Strong NH to NH (i,i+1) nuclear Overhauser enhancements are a feature of the coat protein, which appears to be largely helical. Between 20 and 25 residues give rise to 2 juxtaposed resonances which can be seen clearly in the HMQC spectrum of uniform 15N-labeled coat protein. These residues are concentrated in a region extending from the beginning of the membrane-spanning sequence through to the disordered region near the C-terminus. We propose that dodecyl sulfate-bound M13 coat protein consists of two independent domains, an N-terminal helix which is in a state of moderately fast dynamic flux and a long, stable, C-terminal membrane-spanning helix, which undergoes extensive interactions with a second monomer. Amide 1H chemical shifts are consistent with this picture; in addition, a marked periodicity is observed at the C-terminal end of the molecule.

Amides↗
PubMed1992

The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy.

Previous studies by Wishart et al. [Wishart, D. S., Sykes, B. D., & Richards, F. M. (1991) J. Mol. Biol. (in press)] have demonstrated that 1H NMR chemical shifts are strongly dependent on the character and nature of protein secondary structure. In particular, it has been found that the 1H NMR chemical shift of the alpha-CH proton of all 20 naturally occurring amino acids experiences an upfield shift (with respect to the random coil value) when in a helical configuration and a comparable downfield shift when in a beta-strand extended configuration. On the basis of these observations, a technique is described for rapidly and quantitatively determining the identity, extent, and location of secondary structural elements in proteins based on the simple inspection of the alpha-CH 1H resonance assignments. A number of examples are provided to demonstrate both the simplicity and the accuracy of the technique. This new method is found to be almost as accurate as the more traditional NOE-based methods of determining secondary structure and could prove to be particularly useful in light of the recent development of sequential assignment techniques which are now almost NOE-independent [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667]. We suggest that this new procedure should not necessarily be seen as a substitute to existing rigorous methods for secondary structure determination but, rather, should be viewed as a complement to these approaches.

Amino Acids↗
PubMed1992

Stoichiometry of calcium binding to a synthetic heterodimeric troponin-C domain.

In this work we describe calcium binding to two synthetic 34-residue peptides, determined by 1H-nmr spectroscopy. The peptides investigated, SCIII and SCIV, encompass the calcium-binding sites III and IV, respectively, of troponin-C. In the absence of calcium it has previously been shown that each of these peptides possesses little regular secondary structure. Further, the 1H-nmr spectra of an equimolar mixture of both of these apo-peptides (apo-SCIII/SCIV) shows that little interaction occurs between peptides. Upon calcium binding the spectral changes that occur to SCIII/SCIV are consistent with global conformational changes in both peptides. We have shown previously that these conformational changes are a product of calcium binding to SCIII and SCIV to form a two-site heterodimer Ca2-SCIII/SCIV. It is proposed that this calcium-induced folding proceeds via calcium binding to SCIII to form Ca-SCIII, peptide association with apo-SCIV to form the heterodimer Ca-SCIII/SCIV, and calcium binding to form Ca2-SCIII/SCIV. The dissociation constants involved in this pathway, K1, Kd, and K2, respectively, have been determined by stoichiometric calcium titration of SCIII/SCIV, monitored by 1H-nmr spectroscopy. Using this procedure it has been determined that K1 = 3 microM, Kd = 10 microM, and K2 = 2 microM.

Amino Acid Sequence↗
PubMed1992

Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the alpha-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on alpha-helix formation but resulted in a 100- and 300-fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.

Amino Acid Sequence↗