500-MHz 1H NMR studies of ragweed allergen Ra5

The solution conformation of short ragweed allergen Ra5, a protein of 45 amino acid residues cross-linked with four disulfide bridges, has been investigated by 1H NMR spectroscopy at 500 MHz. The aromatic region, which contains resonances from three tyrosines and two tryptophans, has been partially assigned. Two tyrosines titrate with a pK of 10.2; a third tyrosine is buried under the tryptophan resonances, and its pK could not be determined. The two tryptophans reside in different microenvironments; the resonances of one are very similar to those found in random coil structures while the other has dramatically shifted peaks. Nuclear Overhauser effect (NOE) difference spectroscopy is used to define two distinct spin-diffusion systems for the aromatic residues and to further identify several methyl-containing amino acids involved in these systems. Assignments in the methyl region are based on selective decoupling, chemical shifts, NOE difference spectra, and 2-D J-resolved and 2-D J-correlated spectroscopy (COSY) methodology. A unique ring-current-shifted methyl doublet in the Ra5 spectrum titrates into the bulk methyl region with a pK of 10.2. Examination of the COSY map suggests that this resonance belongs to either leucine-1 or isoleucine-38. Chemical removal of the N-terminal leucine did not affect the ring-current-shifted methyl. Therefore, this unique resonance has been assigned to the methyl of isoleucine-38.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution proton nuclear magnetic resonance studies of the glucocerebrosidase activator protein from Gaucher spleen

A heat-stable protein factor (HSF) obtained from the spleen of a patient with Gaucher's disease that activates glucocerebrosidase was studied by 600-MHz proton NMR spectroscopy. Assignments for a number of aromatic and aliphatic resonances were made on the basis of spin-decoupling, pH-titration, and resolution-enhancement experiments. The upfield ring current shifted aliphatic region and the downfield aromatic region were examined by nuclear Overhauser effect (NOE) methods using both pulsed Fourier-transform spectroscopy and correlation spectroscopy. It was found that a number of upfield-shifted methyl groups and certain methylene groups of specific aliphatic amino acid residues are in proximity relationships with several aromatic residues, forming a compact hydrophobic clustering site. Of special interest, tyrosine A, phenylalanine A, tryptophan B1, and tryptophan B2 were found to be located close to a cluster of aliphatic residues, indicating that the hydrophobic site of the HSF is conformationally rigid and its tertiary structure very compact. A two-dimensional structural model of the hydrophobic site of HSF is proposed.     

Proton resonance assignments of horse ferricytochrome c

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

Determination of local conformational stability in fragment 96-133 of bovine growth hormone by high-resolution H NMR spectroscopy

The specific assignment of resonances in the 400-MHz nuclear magnetic resonance (NMR) spectrum of fragment 96-133 (AII) of bovine growth hormone (bSt) is described. Assignments have been made with homonuclear two-dimensional techniques, in particular that of sequential resonance assignment. Complete assignments were possible for the spin systems of 16 residues out of a total of 38 and partial assignments for another 5. Assignment of resonances to either residue type or a class of residue was possible for a number of other spin systems. Analysis of the type of nuclear Overhauser effect (NOE) indicates that segments 96-110 and 130-133 are nonregular stable structures and that the segment 111-127, which putatively spans the alpha-helix, is not sufficiently stable to generate NOEs.     

Assignment of the aromatic 1H-NMR resonances of myotoxin a isolated from the venom of Crotalus viridis viridis

The 400 MHz 1H-NMR spectrum of myotoxin a from the venom of Crotalus viridis viridis is described. The identification of spin systems in the aromatic region corresponding to the six aromatic residues of myotoxin a was completed using both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts. Assignments of these spin systems to specific residues was possible for the singly occurring amino acids Tyr-1 and Phe-12. Resonances from Tyr-1, His-5 and His-10 were shifted significantly from their random coil values in a pH-dependent manner. These shift perturbations were deemed evidence of a helical arrangement of the amino terminal region which placed these residues in close proximity to each other.     

Specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of the polypeptide cardiac stimulant anthopleurin-A

The specific assignment of resonances in the 300-MHz 1H nuclear magnetic resonance (NMR) spectrum of anthopleurin-A, a polypeptide cardiac stimulant from the sea anemone Anthopleura xanthogrammica, is described. Assignments have been made using two-dimensional NMR techniques, in particular the method of sequential assignments, where through-bond and through-space connectivities to the peptide backbone NH resonances are used to identify the spin systems of residues adjacent in the amino acid sequence. Complete assignments have been made of the resonances from 33 residues out of a total of 49, and partial assignments of a further 3. The resonances from several of the remaining residues have been identified but not yet specifically assigned. A complicating factor in making these assignments is the conformational heterogeneity exhibited by anthopleurin-A in solution. The resonances from a number of amino acid residues in the minor conformer have also been assigned. These assignments contribute towards identification of the origin of this heterogeneity, and permit some preliminary conclusions to be drawn regarding the secondary structure of the polypeptide.     

Sequential 1H NMR assignments and secondary structure of hen egg white lysozyme in solution

Assignments for 1H NMR resonances of 121 of the 129 residues of hen egg white lysozyme have been obtained by sequence-specific methods. Spin systems were identified with phase-sensitive two-dimensional (2-D) correlated spectroscopy and single and double relayed coherence transfer spectroscopy. For key types of amino acid residues, particularly alanine, threonine, valine, and glycine, complete spin systems were identified. For other residues a less complete definition of the spin system was found to be adequate for the purpose of sequential assignment. Sequence-specific assignments were achieved by phase-sensitive 2-D nuclear Overhauser enhancement spectroscopy (NOESY). Exploitation of the wide range of hydrogen exchange rates found in lysozyme was a useful approach to overcoming the problem of spectral overlap. The sequential assignment was built up from 21 peptide segments ranging in length from 2 to 13 residues. The NOESY spectra were also used to provide information about the secondary structure of the protein in solution. Three helical regions and two regions of beta-sheet were identified from the NOESY data; these regions are identical with those found in the X-ray structure of hen lysozyme. Slowly exchanging amides are generally correlated with hydrogen bonding identified in the X-ray structure; a number of exceptions to this general trend were, however, found. The results presented in this paper indicate that highly detailed information can be obtained from 2-D NMR spectra of a protein that is significantly larger than those studied previously.     

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.     

Individual assignments of the methyl resonances in the 1H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor.

In earlier work the resonances of the 20 methyl groups in the basic pancreatic trypsin inhibitor (BPTI) had been identified in the 360-MHz 1H nuclear magnetic resonance (NMR) spectra and most of the methyl lines had from spin-decoupling experiments been assigned to the different types of amino acid residues. The assignments to the different amino acid types were now completed by studies of the saturation transfer between the denatured and the globular forms of the inhibitor and by spin-decoupling experiments in nuclear Overhauser enhancement (NOE) difference spectra. These distinguished between the methyl resonances of Ala and Thr. Furthermore, for most of the methyl resonances, individual assignments to specific residues in the amino acid sequence were obtained from measurements of intramolecular proton-proton NOE's, use of lanthanide NMR shift and relaxation probes, and comparative studies of various chemically modified forms of BPTI. These data provide the basis for individual assignments of the methyl 13C NMR lines in BPTI and for detailed investigations of the relations between the spatial structure of the protein and the chemical shifts of the methyl groups. The methyl groups in BPTI are of particular interest since they are located almost exclusively on the surface of the protein and thus represent potential natural NMR probes for studies of the protein-protein interactions in the complexes formed between BPTI and a variety of proteases.     

A study of the interactions between residues in the C-terminal half of calmodulin by one and two-dimensional NMR methods and computer modelling

Assignments of the six sets of aromatic ring protons and four high-field-shifted methyl group protons of the C-terminal fragment of calmodulin, residues 78-148, was achieved by a combination of one and two-dimensional NMR spectroscopic methods. A full spectral analysis of the aromatic region in terms of chemical shifts and scalar coupling constants was achieved and confirmed by spectral simulation. A three-dimensional structural model of the C-terminal fragment was constructed by interactive computer graphics techniques and combined with nuclear Overhauser enhancements to propose sequence assignments for all aromatic and high-field-shifted methyl groups. This computer-generated three-dimensional model was generally supported by the fact that it qualitatively accounted for many of the ring-current-shifted proton resonances and the intraresidue and interresidue nuclear Overhauser enhancements.     

Proton resonance assignments of horse ferrocytochrome c

Two-dimensional nuclear magnetic resonance (NMR) spectroscopy was used to assign the proton resonances of horse ferrocytochrome c. Assignments were based on the main chain directed (MCD) and sequential assignment procedures. The fundamental units of the MCD approach, the main-chain NH-C alpha H-C beta H J-coupled subspin systems of each amino acid residue (NAB sets), were defined by analysis of direct and relayed coherence transfer spectra. Recognition of main-chain NOE connectivity patterns specified in the MCD algorithm then allowed NAB sets to be aligned in their proper juxtaposition within secondary structural units. The units of secondary structure were placed within the polypeptide sequence of identification of a small number of side-chain J-coupled spin systems, found by direct recognition in 2D spectra of some J-coupled spin systems and by pairwise comparisons of the J-correlated spectra of six homologous cytochromes c having a small number of known amino acid differences. The placement of a given segment in this way defines the amino acid identity of all its NAB sets. This foreknowledge allowed the vast majority of the side-chain resonances to be discerned in J-correlated spectra. Extensive confirmation of the assignments derives internally from multiple main-chain NOE connectivities and their consistency following temperature-induced changes of the chemical shifts of NOE-correlated protons. The observed patterns of main-chain NOEs provide some structural information and suggest small but potentially significant differences between the solution structure observed by NMR and that defined earlier in crystallographic studies at 2.8-A resolution.     

Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation

The solution structure of neuronal bungarotoxin (nBgt) has been studied by using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments for over 95% of the backbone resonances and 85% of the side-chain resonances have been made by using a series of two-dimensional spectra at four temperatures. From these assignments over 75% of the NOESY spectrum has been assigned, which has in turn provided 582 distance constraints. Twenty-seven coupling constants (NH-alpha CH) were determined from the COSY spectra, which have provided dihedral angle constraints. In addition, hydrogen exchange experiments have suggested the probable position of hydrogen bonds. The NOE constraints, dihedral angle constraints, and the rates of amide proton exchange suggest that a triple-stranded antiparallel beta sheet is the major component of secondary structure, which includes 25% of the amino acid residues. A number of NOE peaks were observed that were inconsistent with the antiparallel beta-sheet structure. Because we have confirmed by sedimentation equilibrium that nBgt exists as a dimer, we have reinterpreted these NOE constraints as intermolecular interactions. These constraints suggest that the dimer consists of a six-stranded antiparallel beta sheet (three from each monomer), with residues 55-59 forming the dimer interface.     

Preliminary assignments of the aromatic and some methyl group resonances of the 1H-NMR spectrum of the oxidized form of uteroglobin. Application to the interaction of oxidized uteroglobin with progesterone

Two-dimensional NMR methods have been used to assign aromatic and methyl group resonances in the 1H-NMR spectrum of oxidized uteroglobin. Assignments to specific amino acids are based on X-ray-determined structures of two crystal forms (C222(1) and P2(1] and on an energy-minimized X-ray structure of the C222(1) form of uteroglobin. These preliminary assignments are sufficient to probe the interaction of oxidized uteroglobin with progesterone in solution. The protein global structure is unmodified but some direct or indirect conformational changes are induced in the H1H4(H1'H4') pockets and close to Phe28 by progesterone.     

Two-dimensional 1H-NMR study of the 1-34 fragment of human parathyroid hormone

The complete assignment of resonances in the proton nmr spectrum of the 1-34 amino acid fragment of human parathyroid hormone [hPTH(1-34)], determined using a combination of one- and two-dimensional nmr techniques at 500 MHz, is described. In particular, homonuclear Hartmann-Hahn experiments, recorded in H2O and D2O, are used to resolve ambiguities in the connectivities between the highly overlapped resonances in the aliphatic region of the spectrum. One-dimensional multiple quantum filtering experiments are used to identify serine and phenylalanine spin systems. Analyses of the through-bond and through-space connectivities in the alpha H-NH fingerprint regions of the correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra lead to the assignment of resonances to specific amino acid residues in the polypeptide. Examination of the observed NOE cross peaks indicates that hPTH(1-34) has no detectable secondary structural elements in aqueous solution.     

Computer-aided assignment of the 1H-NMR spectrum of the viral-protein-genome-linked polypeptide from cowpea mosaic virus

The 1H-NMR spectrum of the viral-protein-genome-linked (VPg) polypeptide from cowpea mosaic virus, has been interpreted via application of 2-dimensional (2D) NMR techniques. The interpretation of the data was performed by a computer program called 'PROSPECT' (PROtein SPECTra), which detects the cross-peak patterns of the amino acid residues in the spectra, assigns these patterns to amino acid types, and finally performs the sequential assignments using the well-known 'sequential walks' obtained from the NOE spectrum. Due to the severe overlap of resonances in the NMR spectrum of the VPg polypeptide, several ways of performing these walks existed. The program detected six alternatives for the sequential assignments of backbone N alpha H-C beta H-C beta H moieties.     

The sequence-specific assignment of the 1H-NMR spectrum of an enzyme, horse-muscle acylphosphatase

A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the identification of the main-chain alpha and amide proton resonances. Several of the completely assigned spin systems were then identified as being part of the secondary structure units which led, after analysis of the primary amino acid sequence, to unambiguous sequence-specific assignments. The identification and assignment of the remaining side-chain resonances was then completed and are reported here. These results provide a complete data base for the three-dimensional structure determination of this enzyme in solution.     

Study of angiotensin-(1-7) vasoactive peptide and its beta-cyclodextrin inclusion complexes: complete sequence-specific NMR assignments and structural studies

We report the complete sequence-specific hydrogen NMR assignments of vasoactive peptide angiotensin-(1-7) (Ang-(1-7)). Assignments of the majority of the resonances were accomplished by COSY, TOCSY, and ROESY peak coordinates at 400MHz and 600MHz. Long-side-chain amino acid spin system identification was facilitated by long-range coherence transfer experiments (TOCSY). Problems with overlapped resonance signals were solved by analysis of heteronuclear 2D experiments (HSQC and HMBC). Nuclear Overhauser effects (NOE) results were used to probe peptide conformation. We show that the inclusion of the angiotensin-(1-7) tyrosine residue is favored in inclusion complexes with beta-cyclodextrin. QM/MM simulations at the DFTB/UFF level confirm the experimental NMR findings and provide detailed structural information on these compounds in aqueous solution.