Biomedical subjects

B D Sykes

Publications and source records attributed to B D Sykes.

At least 163 records · Page 9Linked to original sources

PubMed1992

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.

Amino Acid Sequence↗

PubMed1991

Different interactions of Cro repressor dimer with the left and right halves of OR3 operator DNA.

lambda Cro repressor protein is titrated with two half-operator DNA duplexes comprising the right and left halves of the major binding site on phage lambda DNA, the OR3 operator. The comparison of binding strengths and the conformation of Cro repressor in the two protein-DNA complexes shows that base pair differences between the two halves of the OR3 operator affect the binding of Cro repressor protein. Some 1H NMR resonances are assigned for both protein and DNA in the Cro-operator DNA complexes which are then used to highlight differences between Cro right half and Cro left half protein-operator DNA interactions. These differences are compared to the asymmetry found in the lambda C1 repressor-operator DNA complex. Mechanisms for the recognition of the Cro transcriptional regulatory protein have considered only interactions between a single Cro monomer and a consensus half-operator site with the assumption that the interactions in the remaining half-site are related by the 2-fold symmetry of the complex. A revised model is suggested that allows asymmetry in the two halves of the protein-DNA complex. Methods are proposed to avoid problems in the general use of 1H NMR spectroscopy to study protein-DNA interaction such as intermediate exchange behavior and sample aggregation.

Amino Acid Sequence↗

PubMed1991

Relationship between nuclear magnetic resonance chemical shift and protein secondary structure.

An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships. Similar studies, performed on smaller databases, for 13C and 15N chemical shifts reveal equally strong correlations to protein secondary structure. Among the more interesting results to emerge from this work is the finding that all 20 naturally occurring amino acids experience a mean alpha-1H upfield shift of 0.39 parts per million (from the random coil value) when placed in a helical configuration. In a like manner, the alpha-1H chemical shift is found to move downfield by an average of 0.37 parts per million when the residue is placed in a beta-strand or extended configuration. Similar changes are also found for amide 1H, carbonyl 13C, alpha-13C and amide 15N chemical shifts. Other relationships between chemical shift and protein conformation are also uncovered; in particular, a correlation between helix dipole effects and amide proton chemical shifts as well as a relationship between alpha-proton chemical shifts and main-chain flexibility. Additionally, useful relationships between alpha-proton chemical shifts and backbone dihedral angles as well as correlations between amide proton chemical shifts and hydrogen bond effects are demonstrated.

Hydrogen Bonding↗

PubMed1991

Interaction of troponin I and troponin C. Use of the two-dimensional nuclear magnetic resonance transferred nuclear Overhauser effect to determine the structure of the inhibitory troponin I peptide when bound to skeletal troponin C.

We have used two-dimensional 1H nuclear magnetic resonance spectroscopy to determine the structure of the synthetic inhibitory peptide N alpha-acetyl TnI(104-115) amide bound to calcium-saturated skeletal troponin C (TnC). Conformational changes in the peptide induced by the formation of the troponin I (TnI) peptide-TnC complex were followed by the study of the transferred nuclear Overhauser effect, a technique that allows one to determine the structure of a ligand bound to a macromolecule. The structure of the bound TnI peptide reveals an amphiphilic alpha-helix, distorted around the two central proline residues. The central bend in the peptide functions to bring the residues on the hydrophobic face into closer proximity with each other, thereby forming a small hydrophobic pocket. The hydrophilic, basic residues extend off the opposite face of the peptide. Hydrophobic surfaces on TnC that become exposed upon binding of calcium are involved in the binding of the TnI peptide, but electrostatic interactions also contribute to the strength of the interaction. The role of amphiphilic helices in the targeting of calcium-binding proteins such as troponin C will be discussed.

Animals↗

PubMed1991

Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy.

Previous work by Wishart et al. (in press) and others [(1989) J. Magn. Reson. 83, 441-449; (1990) J. Magn. Reson. 90, 165-176] has shown a strong tendency for protein secondary structure to be manifested in 1H NMR chemical shifts. Based on these earlier results, two techniques have been developed for the quantification of secondary structure in proteins. Both methods allow for the rapid and accurate determination of the percent content of helix, coil, and beta-strand based on the integration (or peak enumeration) of selected portions of either 1-D or 2-D 1H NMR spectra. These new and very simple procedures have been found to compare quite favorably to other well established techniques for secondary structure determination such as CD, Raman and IR spectroscopy.

Animals↗

PubMed1991

Probing the relationship between alpha-helix formation and calcium affinity in troponin C: 1H NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides.

Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.

Amino Acid Sequence↗

PubMed1991

Effects of internal motions on the development of the two-dimensional transferred nuclear Overhauser effect.

In this paper we address the influence of internal motions on the development of the transferred nuclear Overhauser effect in a ligand undergoing chemical exchange between a free and a bound state. We examine the effects of varying the effective correlation time as well as the motional order parameter for methyl group and phenyl ring rotations in the free and bound ligand conformations. The effect of decreasing the motional order for a proton pair on a methyl group or phenyl ring is to decrease the effective correlation time of the internuclear vector, and thus to decrease the cross-relaxation rate between the proton pair. This functions to dampen the effects of spin diffusion, especially in the bound ligand where cross-relaxation rates are much faster than in the free ligand. The effect of changing the effective correlation time for methyl group motions has little effect on the build-up behaviour of the transferred nuclear Overhauser effect for small values of fraction bound, but a larger effect on how fast it decays. This effect is greater for internal motions in the free peptide than it is for internal motions in the bound peptide.

Ligands↗

PubMed1991

Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C.

We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.

Animals↗

PubMed1990

Spin-echo 1H NMR studies of differential mobility in gizzard myosin and its subfragments.

The unexpectedly narrow resonances in the 1H NMR spectra of gizzard myosin, heavy meromyosin, and subfragment 1 were examined by spin-echo NMR spectroscopy. These resonances originated predominantly in the myosin heads, or subfragment 1 units. Smooth muscle myosin undergoes a dramatic change in hydrodynamic properties and can exist either as a folded (10S) or as an extended (6S) species. Factors that influence this transition, namely, ionic strength and phosphorylation (or thiophosphorylation), were varied in the NMR experiments. T2 relaxation experiments on dephosphorylated myosin indicated several components of different relaxation times that were not influenced by changes in ionic strength. Our experiments focused on the components with longer relaxation times, i.e., corresponding to nuclei with more mobility, and these were observed selectively in a spin-echo experiment. With dephosphorylated myosin and HMM, increases in ionic strength caused an increased intensity in several of the narrower resonances. The ionic strength dependence of these changes paralleled that for the 10S to 6S transition. With thiophosphorylated myosin and HMM, changes in ionic strength also influenced the intensities of the narrower resonances, and in addition changes in the 1H NMR spectrum due to thiophosphorylation were observed. The narrow resonances seen with myosin and HMM were observed with S1, but the spin-echo spectra of S1 were not influenced either by changes in ionic strength or by phosphorylation. These results suggest that a fraction of the 1H resonances in smooth muscle myosin and its fragments originates from both aliphatic and aromatic residues of increased mobility compared to the mobility expected from hydrodynamic properties of these proteins. In general, the intensities of these residues increase with increasing ionic strength, and this is consistent with an increase in the percentage of mobile residues during the 10S to 6S transition. Segmental flexibility appeared also to be influenced by phosphorylation within the 6S conformation. These changes were not detected in the isolated myosin heads and thus required a higher order of structure, either the subfragment 2 region or the interaction of myosin heads.

Animals↗

PubMed1990

Solution conformation of purine-pyrimidine DNA octamers using nuclear magnetic resonance, restrained molecular dynamics and NOE-based refinement.

The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.

Base Sequence↗

PubMed1990

Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence.

The 70-residue carboxyl-terminal domain of the muscle contractile protein troponin-C contains two helix-loop-helix calcium (Ca)-binding sites that are related to each other by approximate twofold rotational symmetry. Hydrophobic residues from the helices and a short three residue beta sheet at the interface of the two sites act to stabilize the protein domain in the presence of Ca. A synthetic 34-residue peptide representing one of these sites (site III) has been synthesized and studied by H-1 nuclear magnetic resonance (NMR) spectroscopy. In solution this peptide undergoes a Ca-induced conformational change to form the helix-loop-helix Ca-binding motif. Two-dimensional nuclear Overhauser effect spectra have provided evidence for the formation of a beta sheet and interactions between several hydrophobic residues from opposing helices as found in troponin-C. It is proposed that a symmetric two-site dimer similar in tertiary structure to the carboxyl-terminal domain of troponin-C forms from the assembly of two site III peptides in the Ca-bound form.

Amino Acid Sequence↗

PubMed1990

Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: use of the INEPT experiment to follow individual amides in detergent-solubilized M13 coat protein.

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1H nuclear magnetic resonance (NMR) study [O'Neil, J. D. J., & Sykes, B. D. (1988) Biochemistry 27, 2753-2762], multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow "kinetic sets" containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10(5)-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein we use 15N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiment [Morris, G. A., & Freeman, R. (1979) J. Am. Chem. Soc. 101, 760-762] can be used to transfer magnetization to the 15N nucleus from a coupled proton; when 15N-labeled protonated protein is dissolved in 2H2O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H+ and OH- ions. Base catalysis is significantly more effective, resulting in a characteristic minimum rate in model peptides at pH approximately equal to 3. Rate versus pH profiles have been obtained by using the INEPT experiment for the amides of leucine-14, leucine-41, tyrosine-21, tyrosine-24, and valines-29, -30, -31, and -33 in M13 coat protein. The valine residues exchange most slowly and at very similar rates, showing an apparent 10(6)-fold retardation over poly(DL-alanine). A substantial basic shift in the pH of the minimum rate (up to 1.5 pH units) was also observed for some residues. Possible reasons for the shift include accumulation of catalytic H+ ions at the negatively charged micelle surface or destabilization of the negatively charged transition state of the base-catalyzed reaction by either charge or hydrophobic effects within the micelle. The time-dependent exchange-out experiment is suitable for slow exchange rates (kex), i.e., less than (1-2) x 10(-4) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1990

Solution structure of phage lambda half-operator DNA by use of NMR, restrained molecular dynamics, and NOE-based refinement.

The structure of a DNA decamer comprising the left half of the OR3 operator from bacteriophage lambda is determined in solution by using nuclear magnetic resonance spectroscopy and restrained molecular mechanics calculations. Nuclear magnetic resonance assignments for nonexchangeable protons are obtained by two-dimensional correlated and nuclear Overhauser effect (NOE) spectroscopies. Exchangeable proton resonances are assigned by one-dimensional NOE experiments. Coupling constant measurements from one- and two-dimensional experiments are used to determine approximate dihedral angles within the deoxyribose ring. Distances between protons are estimated by extrapolating distances derived from the time-dependent NOE intensities to initial mixing times. The sets of dihedral angles and distances form a basis for structure determination by restrained molecular dynamics. Separate runs start from classical A and from B DNA and converge to essentially identical structures (atomic root mean square difference of 0.8 A). The structures are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated by using a full matrix analysis procedure. Final NOE residual (R) factors were less than 0.19. The resultant structures are generally B type in character, but display local sequence-dependent variations in dihedral angles and in the spatial arrangement of adjacent base pairs. Although the entire structure exhibits a small bend, the central core of the half-operator, which comprises the sequence-specific recognition site for cro repressor, is straight.

Bacteriophage lambda↗

PubMed1990

Detergent-solubilized M13 coat protein exists as an asymmetric dimer. Observation of individual monomers by 15N, 13C and 1H nuclear magnetic resonance spectroscopy.

M13 coat protein is a simple integral membrane protein isolated from the filamentous coliphage M13. Isotopic labels (13C and 15N) may be incorporated biosynthetically into the protein backbone. 13C nuclear magnetic resonance spectroscopy of carbonyl carbon atoms and two-dimensional 1H-detected 15N-1H heteronuclear shift correlation of coat protein in dodecylsulphate micelles have shown many residues throughout the protein to give rise to two distinct resonances of equal intensity. Chemical shift differences between the two forms are small, indicating the existence of two slightly different but equally populated conformational states. We suggest that the two conformers correspond to the inequivalent monomers of an asymmetric coat protein dimer and propose a mechanism for the generation of such a dimer.

Amino Acid Sequence↗

PubMed1990

Structure and dynamics of detergent-solubilized M13 coat protein (an integral membrane protein) determined by 13C and 15N nuclear magnetic resonance spectroscopy.

The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection. M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies. An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments. The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei. Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics. A model is proposed for the structure of a coat protein dimer in detergent micelles.

Amino Acid Sequence↗

PubMed1990

Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments.

This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+X of the coordinating position of the calcium), Asp or Asn at position 3 (+Y), Gly at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix-loop-helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.

Amino Acid Sequence↗

PubMed1989

Site-specific mutants of oncomodulin. 1H NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59----Glu59 substitution in the calcium-specific site.

High resolution 1H nuclear magnetic resonance spectroscopy and optical stopped-flow techniques have been used to study the metal binding properties of a site-specific mutant of bacterial recombinant oncomodulin in which glutamate has replaced a liganding aspartate at position 59 in the CD calcium-binding site. In particular we have followed the replacement of calcium by lutetium in bacterial recombinant oncomodulin and D59E oncomodulin to provide a measure of the protein's preferences for metal ions of different ionic radii. The result of the Asp----Glu substitution is to make the mutant oncomodulin more similar to rat parvalbumin in terms of its relative CD- and EF-domain affinities for lutetium(III), that is to increase its affinity for metal ions with smaller ionic radii. This finding supports the original hypothesis that the presence of Asp at sequence position 59 is an important factor in the reduced preference of the CD site of oncomodulin for smaller metals such as magnesium (Williams, T. C., Corson, D. C., Sykes, B. D., and MacManus, J. P. (1987) J. Biol. Chem. 262, 6248-6256). However, our studies show that both the CD and the EF sites are affected by this single residue substitution suggesting that many factors play a role in the metal binding affinity and interaction between the two sites.

Animals↗

PubMed1989

A 1H NMR determination of the solution conformation of a synthetic peptide analogue of calcium-binding site III of rabbit skeletal troponin C.

NMR techniques have been used to determine the structure in solution of acetyl (Asp 105) skeletal troponin C (103-115) amide, one of a series of synthetic peptide analogues of calcium-binding site III of rabbit skeletal troponin C [Marsden et al. (1988) Biochemistry 27, 4198-4206]. The NMR measurements include 1H-1H nuclear Overhauser enhancements and gadolinium-induced 1H relaxation measurements. The former yield short-range internuclear distances (less than 4 A); the latter, once properly corrected for chemical exchange, yield longer range metal to proton distances (5-10 A). These measurements were then used as pseudo potential energy restraints in energy minimization and molecular dynamics calculations to determine the solution structure. Further information was provided by NMR coupling constants, amide proton exchange rates, and the temperature dependences of amide proton chemical shifts. The solution structure of the peptide analogue is very similar to that of the calcium-binding loop in the protein, the root-mean-square deviation between the backbone atoms being approximately 1.1 A.

Animals↗