PubMed Health⌕ Search

Biomedical subjects

R E Klevit

Publications and source records attributed to R E Klevit.

At least 37 records · Page 2Linked to original sources
PubMed1996

Hydrogen bonding and equilibrium isotope enrichment in histidine-containing proteins.

We have measured deuterium/hydrogen fractionation in three histidine-containing proteins, ecHPr, ecHPr mutant S31A, and bsHPr, and in random coil peptides using NMR and mass spectrometry. The amide protons of unstructured peptides exhibit equilibrium enrichment for deuterium, in agreement with previous studies. Enrichment for both protium and deuterium was observed in both HPrs, with fractionation factors ranging from 0.63 to 1.41. Enrichment for protium was seen in alpha-helical secondary structure. 'Strong' HBs previously identified by mutagenesis and thermodynamic measurements are significantly enriched for protium. Sites of protium enrichment are conserved in a structural context across species lines, though ecHPr and bsHPr share only 30% sequence identity, suggesting that strong HBs are conserved and may play an important role in stabilizing the folded state.

Bacillus subtilis↗
PubMed1995

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

DNA-Binding Proteins↗
PubMed1995

Calcium-dependent solvation of the myristoyl group of recoverin.

Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to function as a calcium sensor in visual signal transduction by coupling the kinetics of the recovery phase of the photoresponse to changes in the levels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed that, in the Ca(2+)-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca(2+)-bound conformation, the myristoyl group is exposed to solution. The crystal structure of Ca(2+)-bound recoverin reveals an exposed cluster of hydrophobic residues, raising the possibility that residues in this region may function as part of an intramolecular myristoyl binding site. Fluorescence spectroscopy analysis of interactions between recoverin and 1-anilinonaphthalene-8-sulfonate (ANS) shows that an increase in solvent-accessible hydrophobic surface accompanies Ca2+ binding. 1H nuclear magnetic resonance (NMR) spectra of myristoyl protons show dispersed chemical shifts in the Ca(2+)-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca(2+)-free recoverin show NOE contacts between myristoyl protons and aromatic ring protons. Tryptophan fluorescence quenching by acrylamide indicates that the myristoyl group is in proximity to a tryptophan residue only in the Ca(2+)-free conformation. These results indicate that the myristoyl group is in contact with residues in the hydrophobic cluster in Ca(2+)-free recoverin and that it is exposed to solution in the Ca(2+)-bound conformation.

Anilino Naphthalenesulfonates↗
PubMed1995

Ca2+ binding to calmodulin and its role in Schizosaccharomyces pombe as revealed by mutagenesis and NMR spectroscopy.

As a first step toward identifying the important structural elements of calmodulin from Schizosaccharomyces pombe, we examined the ability of heterologous calmodulins and Ca(2+)-binding site mutant S. pombe calmodulins to replace the essential cam1+ gene. A cDNA encoding vertebrate calmodulin allows growth of S. pombe. However, calmodulin from Saccharomyces cerevisiae does not support growth even though the protein is produced at high levels. With one exception, all mutant S. pombe calmodulins with one or more intact Ca(2+)-binding sites allow growth at 21 degrees C. A mutant containing only an intact Ca(2+)-binding site 3 fails to support growth, as does S. pombe calmodulin with all four Ca(2+)-binding sites mutated. Several of the mutant proteins confer a temperature-sensitive phenotype. Analysis of the degree of temperature sensitivity allows the Ca(2+)-binding sites to be ranked by their ability to support fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than site 3. A visual colony color screen based on the fission yeast ade1+ gene was developed to perform these genetic analyses. To compare the Ca(2+)-binding properties of individual sites to their functional importance for viability, Ca2+ binding to calmodulin from S. pombe was studied by 1H NMR spectroscopy. NMR analysis indicates a Ca(2+)-binding profile that differs from those previously determined for vertebrate and S. cerevisiae calmodulins. Ca(2+)-binding site 3 has the highest relative affinity for Ca2+, while the affinities of sites 1, 2, and 4 are indistinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca2+ does not predict its functional importance.

Alleles↗
PubMed1995

The Escherichia coli adenylyl cyclase complex: requirement of PTS proteins for stimulation by nucleotides.

GTP, as well as other nucleoside triphosphates, stimulates the activity of Escherichia coli adenylyl cyclase in permeable cells; the stimulatory effect is lost when the cells are disrupted by passage through a French pressure cell. These data suggested that the allosteric regulation by GTP of adenylyl cyclase activity requires an interaction of the enzyme with other protein factors. Strains deleted for genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) failed to show an activity stimulation by GTP. With a view to localizing the site of interaction of GTP with the adenylyl cyclase complex, a variety of studies using purified PTS proteins were performed using the photoaffinity labeling reagent, 8-azidoGTP. These studies showed that 8-azidoGTP bound specifically to HPr. A species specificity study showed that the photoaffinity reagent labeled E. coli HPr but not HPr proteins from Mycoplasma capricolum or Bacillus subtilis. A variety of site-directed mutations of E. coli HPr were evaluated for interaction with GTP by photoaffinity labeling as well as by nuclear magnetic resonance; the results of these studies indicate that the lysine residues at positions 24 and 27, serine-46, the threonine at position 36, and the aspartate at position 69 are important for the binding of GTP to HPr. Molecular modeling has been used to formulate a model for the binding of GTP to HPr involving electrostatic interaction of the phosphate groups of the nucleotide with the side chains of lysine residues 27 and 45 and serine-43, interaction of the sugar with serine-46, and interaction of the base with lysine-24. From these data, it is hypothesized that the binding of GTP to HPr is required for the GTP-dependent stimulation of the activity of the adenylyl cyclase complex.

Adenylyl Cyclases↗
PubMed1995

Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

Phosphorylation of serine-46 in HPr, a key regulatory protein in bacteria, results in stabilization of its solution structure.

The serine-phosphorylated form of histidine-containing protein (HPr), a component of the phosphoenolpyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy and solvent denaturation studies. The results indicate that phosphorylation of Ser 46, the N-cap of alpha-helix-B, does not cause a conformational change but rather stabilizes the helix. Amide proton exchange rates in helix-B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaturation, with a delta delta G of 0.7-0.8 kcal mol-1. A mutant in which Ser 46 is replaced by aspartic acid shows a similar stabilization, indicating that an electrostatic interaction between the negatively charged groups and the helix macrodipole contributes significantly to the stabilization.

Amino Acid Sequence↗
PubMed1994

Structural consequences of histidine phosphorylation: NMR characterization of the phosphohistidine form of histidine-containing protein from Bacillus subtilis and Escherichia coli.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system involves a series of reactions in which phosphoprotein intermediates are formed. Histidine-containing protein (HPr) is phosphorylated on the N delta 1 position of the imidazole ring of His15 by enzyme I and acts as a phosphoryl donor to the sugar-specific enzymes IIA. The structure of phosphorylated HPr from Bacillus subtilis, primarily, and from Escherichia coli has been studied by nuclear magnetic resonance (NMR) spectroscopy. Phosphorylation of His15 results in large downfield shifts in amide proton and nitrogen resonances for residues 16 and 17 but results in only modest or no shifts in other backbone resonances. The exchange rates of these two amide groups are decreased more than 10-fold upon phosphorylation. Analysis of the coupling constants 3JNH alpha revealed no significant changes throughout the protein, indicating that backbone phi dihedral angles do not change detectably. 3J alpha beta and 3JN beta patterns determined from P.E.COSY and HNHB spectra, respectively, revealed a change in one side chain, that of conserved Arg17. Analysis of NOESY spectra revealed a limited number of changes in NOEs involving protons in Ser12, His15, Arg71, and Pro18 in B. subtilis HPr. The NMR results indicate that the Arg17 side chain becomes limited in its conformational range in the phosphorylated protein, taking on a conformation that points its guanidinium group toward the phosphoryl group on His15. In addition, the tautomeric and ionization states of His15 have been determined using 15N and 31P NMR. At neutral pH, the imidazole is predominantly in the protonated form and the phosphoryl group is in the dianionic form in P-His15. Altogether, the results indicate that phosphorylation of His15 yields only a local effect on the protein's structure. The data are consistent with a small change in the disposition of the histidine side chain, allowing phosphoryl group oxygens to serve as hydrogen bond acceptors for the amide protons of residues Ala16 and Arg17, which constitute the first two residues of an alpha-helix. Thus, similar to many proteins that bind phosphoryl moieties noncovalently, the phosphoryl group in P-His15-HPr is situated to allow for a favorable electrostatic interaction at the N-terminal end of an alpha-helix.

Amides↗
PubMed1994

Structure of a histidine-X4-histidine zinc finger domain: insights into ADR1-UAS1 protein-DNA recognition.

The solution structure for a mutant zinc finger peptide based on the sequence of the C-terminal ADR1 finger has been determined by two-dimensional NMR spectroscopy. The mutant peptide, called PAPA, has both proline residues from the wild-type sequence replaced with alanines. A nonessential cysteine was also replaced with alanine. The behavior of PAPA in solution implicates the prolines in the conformational heterogeneity reported earlier for the wild-type peptide [Xu, R. X., Horvath, S. J., & Klevit, R. E. (1991) Biochemistry 30, 3365-3371]. The solution structure of PAPA reveals several interesting features of the zinc finger motif. The residue immediately following the second cysteine ligand adopts a positive phi angle, which we propose is a common feature of this class of zinc fingers, regardless of whether this residue is a glycine. The NMR spectrum and resulting solution structure of PAPA suggest that a side-chain to side-chain hydrogen bond involving an arginine and an aspartic acid analogous to one observed in the Zif268 protein-DNA cocrystal structure exists in solution in the absence of DNA [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. A model for the interaction between the two ADR1 zinc fingers and their DNA binding sites was built by superpositioning the refined solution structures of PAPA and ADR1b onto the Zif268 structure. This model offers structural explanations for a variety of mutations to the ADR1 zinc finger domains that have been shown to affect DNA-binding affinity or specificity.

Alanine↗
PubMed1993

Deamidation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system, involves asparagine 38 (HPr-1) and asparagine 12 (HPr-2) in isoaspartyl acid formation.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

Amino Acid Sequence↗
PubMed1993

The involvement of the arginine 17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.

Amino Acid Sequence↗
PubMed1993

Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae.

The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy. Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified. yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin. As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR. Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM. Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites. Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl). Positive cooperativity was observed among all sites. Ca2+ binding was also monitored indirectly by one-dimensional NMR. Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III). All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2). Thus, an interaction between the two domains of intact yCaM affects the behavior of site III. These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed.

Amides↗
PubMed1993

Structures of DNA-binding mutant zinc finger domains: implications for DNA binding.

Studies of Cys2-His2 zinc finger domains have revealed that the structures of individual finger domains in solution determined by NMR spectroscopy are strikingly similar to the structure of fingers bound to DNA determined by X-ray diffraction. Therefore, detailed structural analyses of single finger domains that contain amino acid substitutions known to affect DNA binding in the whole protein can yield information concerning the structural ramifications of such mutations. We have used this approach to study two mutants in the N-terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc finger sequences spanning residues 102-159. Two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect the DNA-binding activity of ADR1 have previously been identified: H118A and H118Y. The structures of wild-type ADR1b and the two mutant zinc finger domains were determined using two-dimensional nuclear magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated. The molecular architecture of the refined wild-type ADR1b domain is presented in detail. Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild-type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein-DNA interaction.

Amino Acid Sequence↗
PubMed1992

Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD.

The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family. A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells. The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure. There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy. Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity. Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy. Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure. Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer. These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions. Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints. Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer. This topology is hypothesized to serve as a general model for other bHLH protein domains.

Amino Acid Sequence↗
PubMed1992

A series of point mutations reveal interactions between the calcium-binding sites of calmodulin.

Calmodulin is a member of the "EF-hand" family of Ca(2+)-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca(2+)-binding sites. To examine the contribution of individual Ca(2+)-binding sites to the Ca(2+)-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca(2+)-binding loops has been changed to a glutamine. One-dimensional 1H-NMR has been used to monitor Ca(2+)-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca(2+)-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca(2+)-binding loops greatly decreases the Ca(2+)-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca(2+)-binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca(2+)-binding affinities of the partner C-terminal site. In both the N- and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site III or site IV are strikingly different.

Amino Acid Sequence↗
PubMed1992

Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy.

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.

Amino Acid Sequence↗
PubMed1991

Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy.

Analysis of the histidine-containing protein (HPr) from Escherichia coli by two-dimensional homonuclear and heteronuclear nuclear magnetic resonance techniques has been performed, extending the work originally reported [Klevit, R. E., Drobny, G. D., & Waygood, E. B. (1986) Biochemistry 25, 7760-7769; Klevit, R. E., & Drobny, G. P. (1986) Biochemistry 25, 7770-7773; Klevit, R. E., & Waygood, E. B. (1986) Biochemistry 25, 7774-7781]. Two-dimensional homonuclear total coherence spectroscopy (TOCSY) allowed for more complete assignments of the side-chain spin systems than had been possible in the original studies. As well, two-dimensional 15N-1H heteronuclear spectroscopy was used to resolve a number of ambiguities present in the homonuclear spectra due to resonance redundancies. These analyses led to the correction of a number of resonance assignments that were made with the spectra that could be collected with the technology that existed 6 years ago. In addition, amide exchange rates and 3JNH coupling constants have been measured, extending the original analysis and yielding new structural information. All these data have been used to reexamine the folding topology of E. coli HPr. Structure calculations showed that the topology derived from the earlier NMR data, i.e., a four-stranded beta-sheet with three alpha-helices running along one side of the sheet, was essentially unchanged, although at the present level of analysis, a well-defined "helix B" could not be established with high confidence. In addition, the data reported here revealed the existence of two slowly-exchanging side-chain hydroxyl protons belonging to Ser31 and Thr59. Their behavior strongly suggests that these side chains are involved in hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗