Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.     

Native and nonnative conformational preferences in the urea-unfolded state of barstar

The refolding of barstar from its urea-unfolded state has been studied extensively using various spectroscopic probes and real-time NMR, which provide global and residue-specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25 degrees C. Complete backbone resonance assignments of the urea-unfolded protein were obtained using the recently developed three-dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13C(alpha) and 13CO secondary shifts show positive deviations in the urea-unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to beta-strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the beta-strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.     

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.     

NMR structures of salt-refolded forms of the 434-repressor DNA-binding domain in 6 M urea

The N-terminal 63-residue fragment of the phage 434-repressor, 434(1-63), has a well-defined globular fold in H(2)O solution, and is unfolded in 6 M urea at pH 7.5. In this study, 434(1-63) has been refolded by adding either 1.7 M NaCl or 0.47 M NaTFA to the solution in 6 M urea, and the NMR structures of both refolded forms have been determined. The two refolded forms have similar free energies of unfolding and are approximately 16 kJ/mol less stable than the protein in H(2)O solution. 434(1-63) refolded with NaCl exhibits NMR chemical shifts very similar to and a three-dimensional structure nearly identical to those of 434(1-63) in H(2)O solution. The protein refolded with NaTFA also has a similar global fold, but it shows local differences near Phe44, of which two different orientations of the aromatic ring are compatible with the experimental data. This local conformational polymorphism attracted our interest because hydrophobic contacts between two subdomains of residues 1-36 and 45-63 are mediated by the Phe44 side chain. Anion binding experiments suggest that this polymorphism is caused by binding of TFA(-) anions to a cluster of positively charged Arg and Lys residues located in the loop connecting the two subdomains, with apparent binding constants for TFA(-) (K(app)) on the order of 30 mM(-1).     

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.     

Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.     

Structural and dynamic characterization of the acid-unfolded state of hUBF HMG box 1 provides clues for the early events in protein folding

To understand the events that occur in the early stages of the folding of hUBF HMG box 1, we characterized its pH 2.1 unfolded state in detail with NMR. Through a triple resonance strategy, the assignments of complete backbone and some side chains were achieved. Then, significant conformational information was extracted from secondary chemical shifts, interresidual (1)H-(1)H NOEs, (3)J(HNHA) coupling constants, amide proton temperature coefficients, and (15)N relaxation data. The secondary chemical shifts for (13)CA, (13)CB, (13)CO, (1)HA, and (1)HN indicate that the residues between 64 and 78 exhibit a substantial preference for helical structure in the acid-unfolded state, which is also evidenced by the relatively more negative deviations of (3)J(HNHA) and amide proton temperature coefficients from their corresponding random-coil values and particularly confirmed by the strongest sequential d(NN)(i, i + 1) proton NOEs along the region. Following this region until residue 82 is a segment that tends to form a turn-like structure, which is unstable and exchanges between alternative states. In addition, some evidences imply that the regions 18-28 and 38-43 also possess propensities for helical structure but to a different less degree than the region 64-78. The polypeptide backbone dynamics investigated using reduced spectral density function shows apparent motional restrictions in residual structural regions and to less extent at some hydrophobic residues. On the basis of the results presented herein, we propose a potential protein-folding pathway on which these residual structures play a role of initiation site in the early folding stages.     

Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was studied in aqueous solution by nuclear magnetic resonance (NMR). Sequence-specific resonance assignments have been obtained for the complete polypeptide chain of 68 amino acid residues. The secondary structure determined from nuclear Overhauser effects (NOE) and information about slowly exchanging amide protons includes three helical segments consisting of the residues 10-21, 28-38 and 42-52, respectively. Combination of the presently available NMR data with computer modeling provided preliminary evidence for the presence of a helix-turn-helix motif in the homeodomain. Near the turn, this supersecondary structure appears to be very similar to the DNA binding site in the 434 and P22 c2 repressors, but both helices in the homeodomain include 2-3 additional residues when compared with these prokaryotic DNA-binding proteins.     

The application of 1H NMR chemical shift calculations to diastereotopic groups in proteins.

We have calculated chemical shifts for a range of diastereotopic protons in proteins (i.e. methylene protons, and the methyl groups of valine and leucine residues), using a recently optimised method for chemical shift calculation. The calculations are based on crystal structure coordinates, and have been compared with experimental stereospecific assignments. The results indicate that chemical shifts can be used to suggest stereospecific assignments with about 80% probability of being correct, in cases where both the experimental and the calculated chemical shift differences between a pair of diastereotopic protons are greater than 0.3 ppm. Inaccurate calculations are shown to be caused in most cases by differences between crystal and solution structures. Furthermore, chemical shift calculations based on NMR structures are shown to be capable of acting as a further constraint on structure, by limiting the range of side-chain conformations adopted in structures calculated from NMR data.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

Homo- and heteronuclear NMR studies of the human retinoic acid receptor beta DNA-binding domain: sequential assignments and identification of secondary structure elements.

An 80 amino acid polypeptide corresponding to the DNA-binding domain (DBD) of the human retinoic acid receptor beta (hRAR-beta) has been studied by 1H homonuclear and 15N-1H heteronuclear two- and three-dimensional (2D and 3D) NMR spectroscopy. The polypeptide has two putative zinc fingers homologous to those of the receptors for steroid and thyroid hormones and vitamin D3. The backbone 1H resonances as well as over 90% of the side-chain 1H resonances have been assigned by 1H homonuclear 2D techniques except for the three N-terminal residues. The assignments have been confirmed further by means of 15N-1H heteronuclear 3D techniques, which also yielded the assignments of the 15N resonances. Additionally, stereospecific assignments of methyl groups of five valine residues were made. Sequential and medium-range NOE connectivities indicate several elements of secondary structure including two alpha-helices consisting of residues E26-Q37 and Q61-E70, a short antiparallel beta-sheet consisting of residues P7-F9 and S23-C25, four turns consisting of residues P7-V10, I36-N39, D47-C50, and F69-G72, and several regions of extended peptide conformation. Similarly, two helices are found in the glucocorticoid receptor (GR) DBD in solution [H?rd et al. (1990) Science 249, 157-160] and in crystal [Luisi et al. (1991) Nature 352, 497-505], and in the estrogen receptor (ER) DBD in solution [Schwabe et al. (1990) Nature 348, 458-461], although the exact positions and sizes of the helices differ somewhat. Furthermore, long-range NOEs suggest the existence of a hydrophobic core formed by the two helices.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated, tetrameric beta-chain from human adult hemoglobin

The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated beta-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D (1)H NMR in order to assess the extent of assignments allowed by (1)H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the beta-chain to the crystallographically characterized complexes that contains the beta-chain. The target residues are those that exhibit significant (>|0.2| ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 104 target residues ( approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than |0.2| ppm. These comprehensive assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the beta-chain in terms of the crystal coordinates of the beta-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated beta-chain relative to that in the intact R-state HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in the beta-subunit of HbCO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet beta-chain and that this approach should be similarly effective in HbA.     

1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411

The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.     

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.     

A comparative study of dynamic structures between phage 434 Cro and repressor proteins by normal mode analysis

Two DNA binding proteins, Cro and the amino-terminal domain of the repressor of bacteriophage 434 (434 Cro and 434 repressor) that regulate gene expression and contain a helix-turn-helix (HTH) motif responsible for their site-specific DNA recognition adopt very similar three-dimensional structures when compared to each other. To reveal structural differences between these two similar proteins, their dynamic structures, as examined by normal mode analysis, are compared in this paper. Two kinds of structural data, one for the monomer and the other for a complex with DNA, for each protein, are used in the analyses. From a comparison between the monomers it is found that the interactions of Ala-24 in 434 Cro or Val-24 in 434 repressor, both located in the HTH motif, with residues 44, 47, 48, and 51 located in the domain facing the motif, and the interactions between residues 17, 18, 28, and 32, located in the HTH motif, cause significant differences in the correlative motions of these residues. From the comparison between the monomer and the complex with DNA for each protein, it was found that the first helix in the HTH motif is distorted in the complex form. While the residues in the HTH motif in 434 Cro have relatively larger positive correlation coefficients of motions with other residues within the HTH motif, such correlations are not large in the HTH motif of 434 repressor. It is suggestive to their specificity because the 434 repressor is less specific than 434 Cro. Although a structural comparison of proteins has been performed mainly from a static or geometrical point of view, this study demonstrates that the comparison from a dynamic point of view, using the normal mode analysis, is useful and convenient to explore a difference that is difficult to find only from a geometrical point of view, especially for proteins very similar in structure. © 1996 Wiley-Liss, Inc.     

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.     

Sequence-specific 1H NMR assignments and solution structure of bovine pancreatic polypeptide.

Sequence-specific 1H NMR assignments for the 36 residue bovine pancreatic polypeptide (bPP) have been completed. The secondary and tertiary structure of bPP in solution has been determined from experimental NMR data. It is shown that bPP has a very well-defined C-terminal alpha-helix involving residues 15-32. Although regular secondary structure cannot be clearly defined in the N-terminal region, residues 4-8 maintain a rather ordered conformation in solution. This is attributed primarily to the hydrophobic interactions between this region and the C-terminal helix. The two segments of the structure are joined by a turn which is poorly defined. The four end residues both at the N-terminus and the C-terminus are highly disordered in solution. The overall fold of the bPP molecule is very closely similar to that found in the crystal structure of avian pancreatic polypeptide (aPP). The RMS deviation for backbone atoms of residues 4-8 and 15-32 between the bPP mean structure and the aPP crystal structure is 0.65 A, although there is only 39% identity of the residues. Furthermore, the average conformations of some (mostly from the alpha-helix) side chains of bPP in solution are closely similar to those of aPP in the crystal structure. A large number of side chains of bPP, however, show significant conformational averaging in solution.     

Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

Summary Biosynthetically directed fractional incorporation of¹³C into proteins results in nonrandom¹³C-labeling patterns that can be investigated by analysis of the¹³C–¹³C scalar coupling fine structures in heteronuclear¹³C–¹H or homonuclear¹³C–¹³C correlation experiments. Previously this approach was used for obtaining stereospecific¹H and¹³C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the¹H spin-system identifications. Studies of the hydrolysates of fractionally¹³C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of¹³C–¹³C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear¹H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the - and -methyl groups of Ile. In addition to the methyl resonances, the CH₂ groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the -carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.Abbreviations NOE nuclear Overhauser effect - fractional¹³C labeling biosynthetically directed fractional¹³C-labeling - TOCSY total correlation spectroscopy - ROESY rotating frame Overhauser enhancement spectroscopy - [¹³C,¹H]-COSY two-dimensional¹³C–¹H correlation spectroscopy - isotopomer isotope isomer - P22 c2 repressor c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues - P22 c2(1-76) N-terminal domain of the P22 c2 repressor with residues 1–76     

Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.     

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.