Structural comparison of acyl carrier protein in acylated and sulfhydryl forms by two-dimensional 1H NMR spectroscopy

Sequence-specific assignments of 1H NMR resonances are obtained for the backbone protons of Escherichia coli acyl carrier protein, acylated with an eight-carbon chain covalently attached to the prosthetic group thiol (octanoyl-ACP). Comparison of 1H-1H sequential connectivities in the NOESY spectra of octanoyl-ACP and the unacylated protein (ACPSH) indicates that secondary structure is largely conserved on acylation. Changes in resonance positions observed for certain groups of residues are interpreted in terms of a model that describes the spatial reorientation of secondary structural elements in the protein resulting from introduction of the acyl chain.     

Secondary structure of a complement control protein module by two-dimensional 1H NMR

The complement control protein (CCP) module (also known as the short consensus repeat) is a consensus sequence of about 60 amino acid residues which is thought to fold independently. It occurs over 140 times in more than 20 extracellular mosaic proteins including 12 proteins of the complement cascade. An isolated CCP module, the 16th repeat from human complement factor H, has been expressed in a yeast vector and shown to fold with the same pattern of disulfide bond formation as is seen in the native protein. Two-dimensional 600-MHz 1H NMR spectra of this module have been recorded at pH 3.3 and 6.0 and analyzed to permit determination of secondary structure in solution. The CCP module comprises two predominantly extended segments (Glu1-His13 and Ala17-Glu27), two segments of double-stranded antiparallel beta-sheet (Gly14-Val16 paired with Tyr31-Cys33 and Gly38-Asp40 paired with Ser57-Ile59), and a short piece of triple-stranded beta-sheet (Glu27-Thr30, Ile44-Leu48, and Lys51-Ser53). Turns occur at Asp22, Gly36, and Glu50, while Gly41-Ala43 appear to form a looped-out segment or bulge. This structure is compared with a secondary structure prediction made on the basis of an alignment scheme of 101 sequences for CCP modules [Perkins, S. J., Haris, P. I., Sim, R. B., & Chapman, D. (1988) Biochemistry 27, 4004-4012]--the experimentally determined secondary structure bears an overall resemblance to the predicted one but differs in the number and position of turns. Some of those amino acid residues which are highly conserved throughout the range of CCP modules appear to play a role in stabilizing the global fold.     

Secondary structure determination of human beta-endorphin by 1H NMR spectroscopy

The 1H NMR spectra of human beta-endorphin indicate that the peptide exists in random-coil form in aqueous solution but becomes helical in mixed solvent. Thermal denaturation NMR experiments show that in water there is no transition between 24 and 75 degrees C, while a slow noncooperative thermal unfolding is observed in a 60% methanol-40% water mixed solvent in the same temperature range. These findings are consistent with circular dichroism studies by other workers concluding that beta-endorphin is a random coil in water but that it forms 50% alpha-helix or more in mixed solvents. The peptide in the mixed water-methanol solvent was further studied by correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments. These allow a complete set of assignments to be made and establish two distinct stretches over which the solvent induces formation of alpha-helices: the first occurs between Tyr-1 and Thr-12 and the second between Leu-14 and extending to Lys-28. There is evidence that the latter is capped by a turn occurring between Lys-28 and Glu-31. These helices form at the enkephalin receptor binding site, which is at the amino terminus, and at the morphine receptor binding site, located at the carboxyl terminus [Li, C. H. (1982) Cell (Cambridge, Mass.) 31, 504-505]. Our findings suggest that these two receptors may specifically recognize alpha-helices.     

Secondary structure of human granulocyte colony-stimulating factor derived from NMR spectroscopy.

Recombinant 15N-, 13C-labeled human granulocyte colony-stimulating factor (rh-metG-CSF) has been studied by 2D and 3D NMR using uniformly labeled protein as well as residue-specific 15N-labeled samples. Assignment of the 1H, 15N backbone, and 60% 1H sidechain resonances has enabled the determination of the secondary structure of the protein. The secondary structure is dominated by alpha-helical regions with four stretches of helices between residues 11-41, 71-95, 102-124 and 144-170.     

Purification and NMR characterization of acyl carrier protein

The acyl carrier protein preparation obtained using the 2-propanol method of Rock and Cronan (Rock, C. O., and Cronan, J. E., Jr. (1981) Methods Enzymol. 71, 341-351) can be further purified with reversed-phase high-performance liquid chromatography. A homogeneous sample of acyl carrier protein is obtained as determined by NMR and reversed-phase high-performance liquid chromatography.     

Sequence-specific 1H NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional 1H NMR spectroscopy

The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural homology of spinach acyl carrier protein and Escherichia coli acyl carrier protein based on NMR data

Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures. The structure of E. coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792–8797, 1989). Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study. Here we compare this single form to postulated multiple forms of E. coli ACP using the limited amount of NOE data available for the spinach protein. A number of long-range NOE contacts were present between homologous residues in both spinach and E. coli ACP, suggesting tertiary structural homology. To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E. coli ACP. The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E. coli structure, and these were mapped onto the spinach sequence. These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints. All of the experimental spinach NOE constraints could be successfully combined with the E. coli data, confirming the general hypothesis of structural homology. A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case. Proteins 27:131–143 © 1997 Wiley-Liss, Inc.     

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)     

The solution structure of acyl carrier protein from Mycobacterium tuberculosis

Acyl carrier protein (ACP) performs the essential function of shuttling the intermediates between the enzymes that constitute the type II fatty acid synthase system. Mycobacterium tuberculosis is unique in producing extremely long mycolic acids, and tubercular ACP, AcpM, is also unique in possessing a longer carboxyl terminus than other ACPs. We determined the solution structure of AcpM using protein NMR spectroscopy to define the similarities and differences between AcpM and the typical structures. The amino-terminal region of the structure is well defined and consists of four helices arranged in a right-handed bundle held together by interhelical hydrophobic interactions similar to the structures of other bacterial ACPs. The unique carboxyl-terminal extension from helix IV has a "melted down" feature, and the end of the molecule is a random coil. A comparison of the apo- and holo-forms of AcpM revealed that the 4'-phosphopantetheine group oscillates between two states; in one it is bound to a hydrophobic groove on the surface of AcpM, and in another it is solvent-exposed. The similarity between AcpM and other ACPs reveals the conserved structural motif that is recognized by all type II enzymes. However, the function of the coil domain extending from helix IV to the carboxyl terminus remains enigmatic, but its structural characteristics suggest that it may interact with the very long chain intermediates in mycolic acid biosynthesis or control specific protein-protein interactions.     

Malonyl-CoA: acyl carrier protein transacylase from Helicobacter pylori: Crystal structure and its interaction with acyl carrier protein

Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 angstroms resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in alpha/beta hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.     

Improved strategies for the determination of protein structures from NMR data: the solution structure of acyl carrier protein

The hybrid method that combines the early stages of a distance geometry program with simulated annealing in the presence of NMR constraints was optimized to obtain structures fully consistent with the observed NMR data. This was achieved by using more restrictive bounds of the NOE constraints than those usually used in the literature and by grouping the NOEs into classes dependent on the quality of the experimental NOE data. The 'floating' stereospecific assignment introduced at the simulated annealing stage of the calculations further improved the definition of the local conformation. An improved sampling and convergence property of the hybrid method was obtained by means of fitting the substructure obtained from the distance geometry program to different conformations. Compared to the standard hybrid methods, this procedure gave superior structures for a 77 amino acid protein, acyl carrier protein from Escherichia coli.     

The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy.

By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).     

Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Distance constraints from two-dimensional NMR cross-relaxation data are used to derive a three-dimensional structure for acyl carrier protein from Escherichia coli. Several approaches to structure determination are explored. The most successful proves to be an approach that combines the early stages of a distance geometry program with energy minimization in the presence of NMR constraints represented as pseudopotentials. Approximately 450 proton to proton distance constraints including 50 long-range constraints were included in these programs. Starting structures were generated at random by the distance geometry program and energies minimized by a molecular mechanics module to give final structures. Seven of the structures were deemed acceptable on the basis of agreement with experimentally determined distances. Root-mean-square deviations from the mean of these structures for backbone atoms range from 2 to 3 A. All structures show three roughly parallel helices with hydrophobic residues facing inward and hydrophilic residues facing outward. A hydrophobic cleft is recognizable and is identified as a likely site for acyl chain binding.     

The primary structure of spinach acyl carrier protein

Acyl carrier protein (ACP) from spinach leaves has been purified to homogeneity by high-performance liquid chromatography with an anion-exchange column. The amino acid sequence of one major ACP in spinach leaves, ACP-I, has been determined by automated Edman degradation. It consists of the following 82 amino acids: (sequence in text). Sequencing of the intact polypeptide provided data for the first 57 residues. Cleavage of the succinylated ACP with CNBr at Met-46, followed by sequencing of the fragment mixture, provided data for the final 36 residues. The C-terminal alanine was confirmed by carboxypeptidase Y digestion. The spinach ACP has 40, 70, and 25% homology with Escherichia coli, barley, and rabbit ACPs, respectively. The results not only provide the first complete sequence of a plant ACP, but also provide insight into the structural and evolutionary relationships among plant, animal, and bacterial ACPs.     

Secondary and tertiary structure characteristics of Megasphaera elsdenii flavodoxin in the reduced state as determined by two-dimensional 1H NMR

The secondary structure of two-electron-reduced Megasphaera elsdenii flavodoxin has been determined by visual, qualitative inspection of the sequential connectivities involving C alpha H, C beta H and NH protons observed in NOESY (two-dimensional nuclear Overhauser enhancement spectroscopy) spectra. Results from an amide proton exchange experiment were used to confirm the secondary structure assignment and to demonstrate the compactness and stability of the protein. After the secondary structure elements were established, the global fold of the protein and the flavin binding site have been determined using nonsequential interresidual NOE connectivities as primary source of information. The secondary structure and the global fold of M. elsdenii and Clostridium MP flavodoxin appeared to be very similar, differences are observed however. M. elsdenii flavodoxin consists of a central parallel beta-sheet including five strands surrounded on both sides by a pair of alpha-helices.     

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.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

Acyl carrier protein from Escherichia coli. Structural characterization of short-chain acylated acyl carrier proteins by NMR

Acylated acyl carrier proteins (ACPs) with acyl chain lengths of 2, 4, 6, 8, and 10 carbons were investigated by NMR and nuclear Overhauser methods at 500 MHz. Chemical shift changes of downfield aromatic and upfield, ring-current-shifted, isoleucine proton resonances monotonically vary as a function of acyl chain length with the most prominent shifts occurring with chain lengths between four and six carbons. Chemical shifts are largest for one of the two phenylalanines; however, substantial shifts do exist for Tyr-71, His-75, and two isoleucines. Since these residues are distributed throughout the molecule, their associated resonance chemical shifts are most probably explained by an induced conformational change. Comparative NOE measurements on reduced ACP (ACP-SH) and ACP-S-C8 suggest, however, that these induced conformational changes are small except for around one of the phenylalanines. A tertiary structural model for acyl-ACP consistent with our previous model for ACP-SH [Mayo, K. H., Tyrell, P. M., & Prestegard, J. H. (1983) Biochemistry 22, 4485-4493] is presented.     

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.