Identification and description of alpha-helical regions in horse muscle acylphosphatase by 1H nuclear magnetic resonance spectroscopy

It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to locate the two segments within the primary structure (residues 22 to 35 and 55 to 66), thus providing the sequential assignment. The assignment of the side-chains was completed and a list of all nuclear magnetic resonance constraints within the two segments (126 intra- and 180 interresidue distances, 21 torsion angles phi and 19 hydrogen bonds) was produced. Distance geometry calculation shows that each segment forms an alpha-helix. The mutual orientation of the two helices was established subsequently.     

Conformation of the eosinophil chemotactic tetrapeptides and analogues in dimethyl sulfoxide. 1H n.m.r. studies

Proton nuclear magnetic resonance parameters are reported for DMSO-d6 solutions of the eosinophil chemotactic tetrapeptides, Val1-Gly2-Ser3-Glu4 and Ala1-Gly2-Ser3-Glu4, as well as three analogues of the Val1 tetrapeptide, D-Val1, Ala2 and Ala3. The synthesis of Val-(S)-[alpha-2 H1] Gly-Ala-Glu, in which the glycine has been stereospecifically deuterated in the H alpha 3 position, has allowed the assignment of the 1H resonances belonging to individual H alpha 2 and H alpha 3 glycine methylene protons. Simulation of the glycine ABX spin system yields two vicinal coupling constants which are consistent with a highly preferred conformation about the glycine HN-C alpha bond. The chemical shifts, coupling constants, temperature coefficients of amide proton chemical shifts and calculated side chain rotamer populations are reported for all peptides. The coupling constant analysis and temperature coefficients of amide proton chemical shifts together suggest that a type I beta-turn conformation is preferred by the Ala3 analogue. The 1H n.m.r. parameters of the other peptides suggest that these can also adopt a beta-turn conformation in DMSO. There are, however, considerable differences in the extent of conformational averaging undergone by the various peptides.     

Complete covalent structure of nisin Q, new natural nisin variant, containing post-translationally modified amino acids

The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3-7, 8-11, 13-19, 23-26, and 25-28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Basic pancreatic trypsin inhibitor

The assignment of the ¹H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 500 MHz is described. The assignments are based entirely on the known amino acid sequence and the nuclear magnetic resonance data. Individual resonance assignments were obtained for all backbone and C^β protons, with the exception of those of Arg1, Pro2, Pro13 and the amide proton of Gly37. The side-chain resonance assignments are complete, with the exception of Pro2 and Pro13, the N^δ protons of Asn44 and the peripheral protons of the lysine residues and all but two of the arginine residues.     

One- and two-dimensional NMR investigations of the heme pocket in free alpha(CO) chains from human hemoglobin

Two-dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated alpha(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J-correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOSEY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu86, Leu-91, Val-93, Leu-101 and Leu-136. Starting from the known crystallographic structure of the alpha subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring-current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the alpha(CO) subunits in crystals of HbA(CO) and of the free alpha(CO) chains. The one-dimensional NMR spectra were used to monitor the pH-induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p-chloromercuribenzoate is bound to Cys-104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.     

The secondary structure in solution of acyl-coenzyme A binding protein from bovine liver using 1H nuclear magnetic resonance spectroscopy

Acyl-coenzyme A binding protein from bovine liver and the protein expressed in Escherichia coli by the recombinant gene of this protein have been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. This protein has, in addition to the ability to bind acyl-coenzyme A, been reported to have several important physiological and biochemical functions. It is known as the diazepam binding inhibitor, as a putative neurotransmitter, as a regulator of insulin release from pancreatic cells, and as a mediator in corticotropin-dependent adrenal steroidogenesis. The only difference between the protein produced by recombinant techniques and the native acyl-coenzyme A binding protein is the N-terminal acetyl group present only in the native protein. The two proteins have 86 amino acid residues and a molecular mass of approximately 10,000 Da. Complete assignment of the 1H nuclear magnetic resonances has been obtained for a major proportion of the amino acid residues (55 residues), and partial assignment has been achieved for the others (31 residues). Sequential nuclear Overhauser effects have demonstrated that the protein has a secondary structure consisting of four alpha-helices of residues 1-15, 22-35, 52-60, and 68-85. Furthermore, a large number of long-range nuclear Overhauser effects have been identified, indicating that the assignment given here will provide a basis for a structure determination of this protein in solution by nuclear magnetic resonance spectroscopy.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

Assignment of the 1H nuclear magnetic resonance spectrum of the proteinase inhibitor IIA from bull seminal plasma by two-dimensional nuclear magnetic resonance at 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.     

Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.     

Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure

The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Glucagon bound to perdeuterated dodecylphosphocholine micelles

The assignment of the ¹H nuclear magnetic resonance spectrum of glucagon bound to perdeuterated dodecylphosphocholine micelles with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 360 MHz is described. Sequential resonance assignments were obtained for all backbone and C^β protons except the N-terminal amino group and the amide proton of Ser2. The assignments of the non-labile amino acid side-chain protons are complete except for the γ-methylene protons of Gln20 and Gln24. These assignments provide a basis for the determination of the three-dimensional structure of lipid-bound glucagon.     

Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.     

Assignments of backbone 1H, 13C, and 15N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy.

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.     

Solution structure of mitochondrial cytochrome c. I. 1H nuclear magnetic resonance of ferricytochrome c

The 1H nuclear magnetic resonance spectra of tuna and horse ferricytochromes c have been investigated and the resonances of all amino acid methyl groups have been assigned to specific absorption lines. The assignment procedure involves principally the comparison of one-dimensional nuclear magnetic resonance spectra from a range of homologous ferricytochromes c and does not require a prior knowledge of the secondary or tertiary protein structure. Of the 49 methyl groups of tuna cytochrome c, the assignment of 33 is made without reference to the X-ray crystal structure. The method should therefore be applicable to other proteins of similar size where X-ray structures are unavailable. The assignments will be used to investigate the structure of cytochrome c in solution.     

Backbone resonance assignment and order tensor estimation using residual dipolar couplings

An NMR investigation of proteins with known X-ray structures is of interest in a number of endeavors. Performing these studies through nuclear magnetic resonance (NMR) requires the costly step of resonance assignment. The prevalent assignment strategy does not make use of existing structural information and requires uniform isotope labeling. Here we present a rapid and cost-effective method of assigning NMR data to an existing structure—either an X-ray or computationally modeled structure. The presented method, Exhaustively Permuted Assignment of RDCs (EPAR), utilizes unassigned residual dipolar coupling (RDC) data that can easily be obtained by NMR spectroscopy. The algorithm uses only the backbone N–H RDCs from multiple alignment media along with the amino acid type of the RDCs. It is inspired by previous work from Zweckstetter and provides several extensions. We present results on 13 synthetic and experimental datasets from 8 different structures, including two homodimers. Using just two alignment media, EPAR achieves an average assignment accuracy greater than 80%. With three media, the average accuracy is higher than 94%. The algorithm also outputs a prediction of the assignment accuracy, which has a correlation of 0.77 to the true accuracy. This prediction score can be used to establish the needed confidence in assignment accuracy.     

1H NMR studies on bovine cyclophilin: preliminary structural characterization of its complex with cyclosporin A

Cyclophilin (163 residues, Mr 17737), a peptidyl prolyl cis-trans isomerase, is a cytosolic protein that specifically binds the potent immunosuppressant cyclosporin A (CsA). The native form of the major bovine thymus isoform has been analyzed by 2D NMR methods, COSY, HOHAHA, and NOESY, in aqueous media. The 156 main-chain amides in CyP yield 126 observable NH/alpha CH couplings (81%, Gly pairs counted as 1). Following exhaustive D2O exchange, 44 amide resonances remain visible. Further analysis of the NH/NH, NH/alpha CH, and alpha CH/alpha CH regions of the COSY and NOESY data sets indicates that the residual amides in D2O form a coherent hydrophobic domain which yields 2D NMR features suggestive of a beta-sheet. Many (43/126) of the amide resonances have been classified according to amino acid type. In the aromatic region of the spectra, the assignment of the ring spin systems is nearly complete (12/15 Phe, 2/2 Tyr, 1/1 Trp, and 3/4 His). This has successfully lead to the complete assignment of all of their beta CH's, main-chain alpha CH resonances, and many of the backbone amide resonances (8/12 Phe, 2/2 Tyr, 1/1 Trp, and 2/3 His). In other regions of the spectrum, the side-chain and main-chain resonances for 10/23 Gly, 9/9 Ala, 5/11 Thr, 5/9 Val, and 1/6 Leu have been completely assigned. The drug-free cyclophilin and CsA-bound cyclophilin form two discrete protein structures that are in slow exchange on the NMR time scale. Comparison of the fingerprint regions from the COSY spectra obtained from the two forms of the protein reveals a minimum of 16 cross-peaks which are clearly shifted upon complexation. In fact, on the basis of chemical shift changes observed in assigned side-chain and main-chain resonances, only a relatively few of the amino acid residues identified to date are perturbed by complex formation. These include 3 Phe (8, 12, and 14) and the Trp in the aromatic region and 2 Ala (7 and 8) in the Ala/Thr region. In the upfield-shifted methyl region, an assigned Leu and Val spin system and a spin system labeled X10 (an Ile or Leu) are affected by complex formation. In addition, a new aliphatic spin system, labeled X11, which shows a close spatial relationship to the perturbed Phe12, is observed in this region of the spectrum. In summary, the regions of the protein altered by complex formation can be divided into two categories: a hydrophobic and a H2O-accessible domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequential 1H NMR assignments and secondary structure of the B domain of staphylococcal protein A: structural changes between the free B domain in solution and the Fc-bound B domain in crystal

The recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G (IgG), has been investigated with the use of two-dimensional proton nuclear magnetic resonance spectroscopy. All backbone and side-chain proton resonances of FB (60 amino acid residues), except the amide proton resonance of Ala2, were assigned by the sequential assignment procedures by using double-quantum-filtered correlated spectroscopy (DQF-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and nuclear Overhauser enhancement spectroscopy (NOESY). On the basis of the NOESY data, three helical regions, Glu9-His19, Glu25-Asp37, and Ser42-Ala55, were identified in the free FB in solution. Existence of two of the three helical regions, Glu9-His19 and Glu25-Asp37, in consistent with the X-ray crystallographic structure of the Fc-bound FB [Deisenhofer, J. (1981) Biochemistry 20, 2361-2370]. By contrast, in the Fc-bound FB as revealed by the X-ray analysis, the Ser42-Glu48 segment is extended and no structural information has been available in the Ala49-Ala55 segment. We suggest that a significant conformation change is induced in the C-terminal region of FB when it is bound to the Fc portion of IgG.     

A random graph approach to NMR sequential assignment.

Nuclear magnetic resonance (NMR) spectroscopy allows scientists to study protein structure, dynamics and interactions in solution. A necessary first step for such applications is determining the resonance assignment, mapping spectral data to atoms and residues in the primary sequence. Automated resonance assignment algorithms rely on information regarding connectivity (e.g., through-bond atomic interactions) and amino acid type, typically using the former to determine strings of connected residues and the latter to map those strings to positions in the primary sequence. Significant ambiguity exists in both connectivity and amino acid type information. This paper focuses on the information content available in connectivity alone and develops a novel random-graph theoretic framework and algorithm for connectivity-driven NMR sequential assignment. Our random graph model captures the structure of chemical shift degeneracy, a key source of connectivity ambiguity. We then give a simple and natural randomized algorithm for finding optimal assignments as sets of connected fragments in NMR graphs. The algorithm naturally and efficiently reuses substrings while exploring connectivity choices; it overcomes local ambiguity by enforcing global consistency of all choices. By analyzing our algorithm under our random graph model, we show that it can provably tolerate relatively large ambiguity while still giving expected optimal performance in polynomial time. We present results from practical applications of the algorithm to experimental datasets from a variety of proteins and experimental set-ups. We demonstrate that our approach is able to overcome significant noise and local ambiguity in identifying significant fragments of sequential assignments.     

Signal peptide of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.     

Assignment of the 1H nuclear magnetic resonance spectrum of the trypsin inhibitor homologue K from Dendroaspis polylepis polylepis. Two-dimensional nuclear magnetic resonance at 360 and 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the trypsin inhibitor homologue K from the venom of Dendroaspis polylepis polylepis is described and documented. The assignments are based entirely on the amino acid sequence and on 2-dimensional n.m.r. experiments at 360 and 500 M Hz. Individual assignments were obtained for the backbone and C beta protons of all 57 residues of the inhibitor homologue K, with the exceptions of the N-terminal amino group, the amide protons of Arg16, Gly37 and Gly40 and the C beta protons of Arg16 and Pro19. The assignments for the non-labile protons of the amino acid side-chains are complete, with the exception of Gln29, Glu49 and all the proline, lysine and arginine residues. For Asn and Trp the labile side-chain protons have also been assigned. The chemical shifts for the assigned resonances are listed for an aqueous solution at 50 degrees C and pH 3.4.