PubMed Health⌕ Search

Biomedical subjects

R E Klevit

Publications and source records attributed to R E Klevit.

67 records · Page 4Linked to original sources
PubMed1986

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 2. Leucine resonance assignments by long-range coherence transfer.

Sequence-specific assignments of the NH, C alpha H, and C beta H resonances in the NMR spectrum of the histidine-containing protein (HPr) from Escherichia coli are complete [Klevit, R. E., Drobny, G. P., & Waygood, E. B. (1986) Biochemistry (first paper of three in this issue)]. In addition, the C gamma H3 resonances of valyl, threonyl, and isoleucyl residues have been assigned by two-dimensional relayed coherence transfer (RELAY) experiments. In order to rigorously assign the resonances from longer side chains such as leucines, long-range transfer experiments have been applied to HPr. Coherence transfers via isotropic mixing within large spin systems were accomplished by multiple pulse trains applied during the mixing time of a two-dimensional experiment.

Bacterial Proteins↗
PubMed1986

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 3. Secondary and tertiary structure as determined by NMR.

Sequence-specific resonance assignments of the 1H NMR spectrum of the 85-residue histidine-containing phosphocarrier protein (HPr) are complete [Klevit, R. E., Drobny, G. P., & Waygood, E. B. (1986) Biochemistry (first paper of three in this issue)]. Additional side-chain assignments have been made with long-range coherence transfer experiments [Klevit, R. E., & Drobny, G. P. (1986) Biochemistry (second paper of three in this issue)]. In this paper, the NMR assignments were used to determine the secondary structure and the tertiary folding of HPr in solution. The secondary structural elements of the protein were determined by visual inspection of the pattern of nearest-neighbor nuclear Overhauser effects (NOEs) and the presence of persistent amide resonances. Escherichia coli HPr consists of four beta-strands, three alpha-helices, four reverse turns, and several regions of extended backbone structure. Long-range NOEs, especially among side-chain protons, were used to determine the tertiary structure of the protein by use of the secondary structural components. The four beta-strands form a single antiparallel beta-pleated sheet. The hydrophobic faces of the alpha-helices interact to form a hydrophobic core and sit above the hydrophobic face of the beta-sheet, forming an open-face beta-sheet sandwich structure. The active site histidine, His-15, is on a short kinked segment of backbone that is accessible to the solvent. The positively charged phosphorylation site (His-15 and Arg-17) interacts with the negatively charged carboxyl terminus of the protein (Glu-85).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

1H NMR studies on the interaction between distamycin A and a symmetrical DNA dodecamer.

High-resolution NMR techniques have been used to examine the structural and dynamical features of the interaction between distamycin A and the self-complementary DNA dodecamer duplex d-(CGCGAATTCGCG)2. The proton resonances of d(CGCGAATTCGCG)2 have been completely assigned by previous two-dimensional NMR studies [Hare, D. R., Wemmer, D. E., Chou, S. H., Drobny, G., & Reid, B. R. (1983) J. Mol. Biol. 171, 319-336]. Addition of the asymmetric drug molecule to the symmetric dodecamer leads to the formation of an asymmetric complex as evidenced by a doubling of DNA resonances over much of the spectrum. In two-dimensional exchange experiments, strong cross-peaks were observed between uncomplexed DNA and drug-bound DNA resonances, permitting direct assignment of many drug-bound DNA resonances from previously assigned free DNA resonances. Weaker exchange cross-peaks between formerly symmetry related DNA resonances indicate that the drug molecule flips head-to-tail on one duplex with half the frequency at which it leaves the DNA molecule completely. In experiments performed in H2O, nuclear Overhauser effects (NOEs) were observed from each drug amide proton to an adenine C2H and a pyrrole H3 ring proton. In two-dimensional nuclear Overhauser experiments performed on D2O solutions, strong intermolecular NOEs were observed between each of the three pyrrole H3 resonances of the drug and an adenine C2H resonance, with weaker NOEs observed between the drug H3 resonances and C1'H resonances. The combined NOE data allow us to position the distamycin A unambiguously on the DNA dodecamer, with the drug spanning the central AATT segment in the minor groove.

Base Sequence↗
PubMed1985

Interaction of calmodulin and a calmodulin-binding peptide from myosin light chain kinase: major spectral changes in both occur as the result of complex formation.

Many different enzymes are activated by direct interaction with calmodulin; this interaction is thought to occur through a distinct calmodulin-binding domain in each of these enzymes. We have recently reported the sequence of a 27-residue peptide (denoted M13), derived from skeletal muscle myosin light chain kinase (MLCK), that exhibits the properties expected of a calmodulin-binding domain [Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191]. The interaction between chemically synthesized M13 peptide and calmodulin has been studied by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) spectroscopy. In the presence of Ca2+, the observed ellipticity of an equimolar mixture of M13 and calmodulin is much greater than the sum of the ellipticities of the two isolated proteins. In the absence of Ca2+, the measured ellipticity of the mixture is approximately the sum of the two components. Addition of the peptide to calmodulin causes dramatic changes in the proton NMR spectrum; at a 1:1 molar ratio, no evidence of either free peptide or free calmodulin is observed. Moreover, these data demonstrate that a unique species of the M13-calmodulin complex is formed, indicating that the peptide binds to calmodulin in only one way. The many resonances affected by M13 binding include residues in both halves of the calmodulin molecule. The observed CD and NMR effects suggest that secondary and tertiary conformational changes occur both in M13 and in calmodulin upon complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Azidotyrosylcalmodulin derivatives. Specific probes for protein-binding domains.

Azidocalmodulin has been shown to be a useful probe for calmodulin-binding proteins in a variety of systems (Andreasen, T. J., Keller, C. H. LaPorte, D. C., Edelman, A. M., and Storm, D. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2782-2785). In previous work, this calmodulin derivative was generated by the modification of lysyl residues. We report here that, on the basis of tryptic peptide mapping by reverse-phase high-pressure liquid chromatography, azidolysylcalmodulin prepared by these procedures is modified at 4 of the 7 lysines. We also report the preparation of azidocalmodulins in which the photolabile moiety is incorporated into a single known residue of the molecule by modifying one or the other of the two tyrosyl side chains. This yields azido-Tyr 99-calmodulin, with the photoaffinity label in Ca2+-binding loop III, and azido-Tyr 138-calmodulin, with the photoaffinity label in Ca2+-binding loop IV. The cross-linking characteristics of these two calmodulin derivatives show that the formation of a covalent adduct upon photolysis of a mixture of azidocalmodulin and a target protein is dependent on the location of the nitrene generated by the irradiation. Azido-Tyr 138-calmodulin shows a significant decrease in cross-linking efficiency to targets such as troponin I, troponin T, and myosin light-chain kinase, relative to azido-Tyr 99-calmodulin and the azidolysyl derivatives.

Amino Acids↗
PubMed1984

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.

Animals↗
PubMed1984

Heterogeneity of contractile proteins. Purification and characterization of two species of troponin T from rabbit fast skeletal muscle.

Two species of troponin T have been purified by ion-exchange chromatography from erector spinae, the major fast white muscle of the rabbit back, and from a pool of the fast hindlimb muscles gastrocnemius and plantaris. Designated Tn-T1f and Tn-T2f, they can be resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, with apparent molecular weights of 37,500 and 37,000 respectively. Their amino acid compositions are similar and correlate well with that reported for troponin T from fast muscle (Pearlstone, J. R., Carpenter, M. R., and Smillie, L. B. (1977) J. Biol. Chem. 252, 971-977). Tn-T2f most likely corresponds to the previously studied troponin T; further characterization was undertaken to determine how the newly identified Tn-T1f differs from Tn-T2f. Phosphorylation of alkaline phosphatase-treated troponin demonstrated that Tn-T1f and Tn-T2f are not interconverted by a change in phosphorylation state. Comparison of the CNBr fragments of Tn-T1f and Tn-T2f by SDS-gel electrophoresis and reverse phase high-performance liquid chromatography revealed similar but not identical peptide patterns. The major difference occurs in the amino-terminal CNBr peptides corresponding to CB3. Since both Tn-T1f and Tn-T2f have blocked amino termini, the difference does not result from proteolysis at the amino terminus of one of the proteins. These observations indicate that the two species of troponin T do not result from a known post-translational modification, but rather from differences in the amino acid sequence, suggesting that they arise either from the expression of different genes or a single gene from which different mRNAs are transcribed.

Amino Acids↗
PubMed1984

1H-NMR studies of calmodulin. The nature of the Ca2+-dependent conformational change.

Using assignments of resonances in the 1H-NMR spectrum of calmodulin obtained by the use of large tryptic fragments of the molecule [Dalgarno, D. C., Klevit, R. E., Levine, B. A., Williams, R. J. P., Dobrowolski, Z., and Drabikowski, W. (1984) Eur. J. Biochem. 138, 281--289], the spectral changes which occur on Ca2+ binding to calmodulin have been examined in detail. Ca2+ binding occurs in two stages: the first two Ca2+ ions bind at sites III and IV (numbered from the N terminus) and the second two Ca2+ ions bind at sites I and II. The high-affinity binding causes perturbations of residues in both halves of the molecule, whereas binding at the two N-terminal sites only affects sidechains in that half of the molecule. The effects of binding Cd2+ to the Ca2+-binding sites of calmodulin have also been studied by 1H NMR. The cation induces spectral changes which are very similar to those seen for Ca2+, but some important differences do exist.

Binding Sites↗
PubMed1984

1H NMR studies of calmodulin. Resonance assignments by use of tryptic fragments.

Two tryptic fragments of the Ca2+ -binding protein calmodulin have been studied by high-resolution 1H NMR. TR1C (residues 1 - 77) spans the first two domains of the protein and TR2C (residues 78 - 148) spans the second two domains. The spectra indicate that each of the two-domain peptides assumes a conformation which is very close to that in the native protein. This characteristic holds both in the presence and in the absence of Ca2+ ions. Therefore, the resonance assignments obtained for the relatively simpler fragment spectra can be used to assign the spectrum of whole calmodulin. Analysis of the chemical shift patterns and nuclear Overhauser enhancement effects of several assigned resonances indicates that each half of calmodulin can be modelled after the two EF-hand Ca2+-binding proteins for which crystal structures are available, namely parvalbumin and intestinal Ca2+-binding protein.

Animals↗
PubMed1983

The mobility of calcium-trigger proteins and its function.

Trigger activity implies the transfer of the energy of a signal to some amplified (energy) response. Actions in cells, from calcium concentration changes to major protein reorganization are discussed here. The changes must be fast, so mobile polymers must be involved. The first step is the calcium on/off binding to its receptor, calmodulin, troponin C or a comparable protein. Calcium binding is to a loop, EF-hand, between helices. The structures and internal mobilities of these proteins are described using nuclear magnetic resonance and the temperature dependence of NMR shifts. It is suggested that these proteins illustrate a general working hypothesis that proteins made from interacting helices as opposed to beta-sheet proteins will have relatively easy internal main chain motions. Loops connecting the helices then provide particularly obvious read-out points, for example of the initial message of calcium binding. These and other regions of loose structure appear to be associated with highly charged sequences. The further transfer of the trigger message is to highly mobile sequences in troponin I, troponin T and tropomyosin.

Amino Acid Sequence↗