Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein.

The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys-Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an alphahelix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain chi1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.     

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.     

Dynamics of a de novo designed three-helix bundle protein studied by 15N, 13C, and 2H NMR relaxation methods

Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. Alpha3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of alpha3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, alpha3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of alpha3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13C(alpha) relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the alpha-helical regions of alpha3D, with increasing mobility at the ends of the alpha-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of alpha3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and beta-branched amino acids. The time scale of motions associated with methyl-bearing side chains of alpha3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of alpha3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.     

The unusual internal motion of the villin headpiece subdomain

The thermostable 36‐residue subdomain of the villin headpiece (HP36) is the smallest known cooperatively folding protein. Although the folding and internal dynamics of HP36 and close variants have been extensively studied, there has not been a comprehensive investigation of side‐chain motion in this protein. Here, the fast motion of methyl‐bearing amino acid side chains is explored over a range of temperatures using site‐resolved solution nuclear magnetic resonance deuterium relaxation. The squared generalized order parameters of methyl groups extensively spatially segregate according to motional classes. This has not been observed before in any protein studied using this methodology. The class segregation is preserved from 275 to 305 K. Motions detected in Helix 3 suggest a fast timescale of conformational heterogeneity that has not been previously observed but is consistent with a range of folding and dynamics studies. Finally, a comparison between the order parameters in solution with previous results based on solid‐state nuclear magnetic resonance deuterium line shape analysis of HP36 in partially hydrated powders shows a clear disagreement for half of the sites. This result has significant implications for the interpretation of data derived from a variety of approaches that rely on partially hydrated protein samples.     

Exposed tyrosine residues of lambda cro repressor protein evidenced by nitration and photo CIDNP experiments

The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).     

Thermodynamic genetics of the folding of the B1 immunoglobulin-binding domain from streptococcal protein G

A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: (1) those that disrupt packing of hydrophobic side chains in the protein interior; (2) those that destabilize secondary structure; and (3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2–5 kcal/mol. © 1995 Wiley-Liss, Inc.     

Role of hydrophobic amino acids in gurmarin,a sweetness-suppressing polypeptide

The sweetness-suppressing polypeptide gurmarin isolated from Gymnema sylvestre consists of 35 amino acid residues and contains three intramolecular disulfide bonds. Nuclear magnetic resonance analysis showed that the hydrophobic side chains of Tyr-13, Tyr-14, Trp-28, and Trp-29 in gurmarin are oriented outwardly. Together with the hydrophobic side chains of Leu-9, Ile-11, and Pro-12, they form a hydrophobic cluster, and therefore these hydrophobic groups are assumed to act as the site for interaction with the receptor protein. To examine the roles of these hydrophobic amino acids, they were replaced by Gly. The resulting [Gly^13,14,28,29]gurmarin and [Gly^{9,11,13,14,28,29}]gurmarin did not suppress the responses to sucrose, glucose, fructose, or Gly. This result strongly suggests that these hydrophobic amino acids are involved in the interaction with the receptor protein. © 1998 John Wiley & Sons, Inc. Biopoly 45: 231–238, 1998     

Solution structure of Phl p 3, a major allergen from timothy grass pollen

The major 97-aa timothy grass (Phleum pratense) allergen Phl p 3 was recently isolated from an extract of timothy grass pollen. Sequence comparison classifies this protein as a group 3 allergen. The solution structure of Phl p 3 as determined by nuclear magnetic resonance spectroscopy reveals that the protein consists of a core of hydrophobic amino-acid side chains from two beta-sheets of five and four anti-parallel beta-strands, respectively. This conformation is very similar to the crystal structure published for Phl p 2 and strongly resembles the known conformation of the carboxy-terminal domain of Phl p 1, the major difference being the loop orientations. Phl p 2 and Phl p 3 show virtually identical immunoreactivity, and comparison of the charged surface amino acids of the two proteins gives initial clues as to the IgE recognition epitopes of these proteins.     

Formation of fumonisin artefacts in thermal treated food

In order to study the formation of fumonisin artefacts and the binding of fumonisins to matrix components (e.g. starch and protein) in thermal treated food, model experiments were performed by heating fumonisin FB₁ with amino acid derivatives (protein model) and methyl-a-D-glucopyranoside (starch model). The reaction products were analysed by highperformance liquid chromatography-electrospray ionisationtandem mass spectrometry (HPLC-MS/MS). Using MS/MS experiments the formed reaction products were characterized as conjugates between fumonisin B₁ and the used substrates. The reaction product between fumonisin B₁ and methyl-α-D-glucopyranoside was purified and identified by nuclear magnetic resonance (NMR) spectroscopy as the diester of fumonisin B₁ and methyl-α-D-glucopyranoside. These studies indicate that fumonisins can bind to matrix components via their TCA side chains.     

Photochemical surface mapping of C14S-Sml1p for constrained computational modeling of protein structure

Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server. The top five models initially generated by the server all disagreed with both published nuclear magnetic resonance (NMR) data and the solvent-accessibility data obtained in this study. A structural model adjusted to fit the previously reported NMR data satisfied most of the solvent-accessibility constraints. Through minor adjustment of the rotamers of two amino acid side chains for this latter structure, a model that not only provided a lower energy conformation but also completely satisfied previously reported data from NMR and tryptophan fluorescence measurements, in addition to the solvent-accessibility data presented here, was generated.     

Folding determinants of disulfide bond forming protein B explored by solution nuclear magnetic resonance spectroscopy

The two-stage model for membrane protein folding postulates that individual helices form first and are subsequently packed against each other. To probe the two-stage model, the structures of peptides representing individual transmembrane helices of the disulfide bond forming protein B have been studied in trifluoroethanol solution as well as in detergent micelles using nuclear magnetic resonance (NMR) and circular dichroism spectroscopy. In TFE solution, peptides showed well-defined α-helical structures. Peptide structures in TFE were compared to the structures of full-length protein obtained by X-ray crystallography and NMR. The extent of α-helical secondary structure coincided well, lending support for the two-stage model for membrane protein folding. However, the conformation of some amino acid side chains differs between the structures of peptide and full-length protein. In micellar solution, the peptides also adopted a helical structure, albeit of reduced definition. Using measurements of paramagnetic relaxation enhancement, peptides were confirmed to be embedded in micelles. These observations may indicate that in the native protein, tertiary interactions additionally stabilize the secondary structure of the individual transmembrane helices.     

Sequence-specific 1H NMR assignments and identification of slowly exchanging amide protons in murine epidermal growth factor

Proton nuclear magnetic resonance (1H NMR) assignments for the murine epidermal growth factor (mEGF) in aqueous solution were determined by using two-dimensional NMR at pH 3.1 and 28 degrees C. The assignments are complete for all backbone hydrogen atoms, with the exception of the N-terminal amino group, and for 46 of the 53 side chains. Among the additional seven amino acid residues, three have complete assignments for all but one side-chain proton, and between two and four protons are missing for the remaining four residues. The sequential assignments by nuclear Overhauser effect spectroscopy are consistent with the chemically determined amino acid sequence. The NMR data show that the conformations of both the Tyr3-Pro4 and Cys6-Pro7 peptide bonds are trans in the predominant solution structure. Proton-deuterium exchange rate constants were also measured for 13 slowly exchanging amide protons. The information presented here has been used elsewhere to determine the three-dimensional structure of mEGF in aqueous solution.     

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.     

Assignment of proton resonances in the NMR spectrum of carbonmonoxy hemoglobin beta subunit tetramers

Isolated beta chains from human adult hemoglobin at millimolar concentration are mainly associated to form beta 4 tetramers. We were able to obtain relevant two-dimensional proton nuclear magnetic resonance (NMR) spectra of such supermolecular complexes (Mr approximately 66,000) in the carboxylated state. Analysis of the spectra enabled us to assign the major part of the proton resonances corresponding to the heme substituents. We also report assignments of proton resonances originating from 12 amino acid side chains mainly situated in the heme pocket. These results provide a basis for a comparative analysis of the tertiary heme structure in isolated beta(CO) chains in solution and in beta(CO) subunits of hemoglobin crystals. The two structures are generally similar. A significantly different position, closer to the heme center, is predicted by the NMR for Leu-141 (H19) in isolated beta chains. Comparison of the assigned resonances of conserved amino acids in alpha chains, beta chains and sperm whale myoglobin indicates a close similarity of the tertiary heme pocket structure in the three homologous proteins. Significant differences were noted on the distal heme side, at the position of Val-E11, and on Leu-H19 and Phe-G5 position on the proximal side.     

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

Background  

The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction.     

A nuclear magnetic resonance study of the helix–coil transition of poly(L-glutamic acid)

There have been many reports that the nuclear magnetic resonance (nmr) spectra of a large number of polypeptides exhibit peak doubling of the α-carbon and the α-carbon proton in the helix–coil transition region. One apparent exception to this generalization has been polypeptides with ionizable side chains, where the helix–coil transition is induced by changes in pH in aqueous solution. Because it is important to establish the proper theoretical reason for the peak doubling and its relation to the rate of conformational change of amino acid residues, we have reexamined the proton and carbon-13 nmr spectra, at high field, for two polydisperse samples of poly(L -glutamic acid). Doubling of the α-carbon proton resonance as well as those of the α- and β-carbon, and backbone carbonyl are observed for a low-molecular-weight sample (DP = 54), while a higher molecular weight sample (DP = 309), exhibits only single resonances. Thus, polydispersity by itself is not sufficient to observe peak doubling; low-molecular weight is also required.     

The use of p-fluorobenzenesulfonyl chloride as a reagent for studies of proteins by fluorine nuclear magnetic resonance

The reagent p-fluorobenzenesulfonyl chloride modifies the protein side chains of tyrosine, lysine, and histidine and the alpha-NH2 group. The p-fluorobenzenesulfonyl (Fbs-) group, identified by the 19F nuclear magnetic resonance signal, exhibits a different 19F chemical shift for each functional group modified. The Fourier-transformed spectra of the Fbs- group displayed the expected nine-line multiplet in Fbs- amino acids and simple Fbs- peptides but not in the Fbs- proteins, where the resolution was less. Lysozyme, RNase, DNase, and chymotrypsin react with this reagent and each Fbs- protein exhibits a distinctive pattern of 19F NMR signals due to the label, suggesting that the reaction of the reagent varies with the reactivity of the side chains in a protein. The three major 19F signals of the unfolded Fbs-RNase in 8 M urea are due to the Fbs- label on the imidazolium, alpha-NH2, and epsilon-NH2 groups. Based upon results from amino acid and 19F NMR analyses of the tryptic-chymotryptic peptides of Fbs-RNase, portions of the imidazolium and epsilon-NH2 resonances were assigned to the Fbs- label on His-105 and Lys-41, respectively, while the alpha-NH2 resonance was entirely due to the Fbs- label on the alpha-NH2 of Lys-1. Because Fbs-RNase has an unchanged, near-ultraviolet circular dichroism spectrum and because it retains approximately 80% of the RNase activity, the conformation of Fbs-RNase is probably not altered from the folded conformation of the native enzyme. Upon unfolding in 8 M urea or heating at 70 degrees C, Fbs-RNase gave a 19F NMR spectrum differing from that of the folded Fbs-RNase. In the presence of uridylic acid, Lys-41 was the only residue protected from modification by the reagent with a concomitant reduction of the epsilon-NH2 resonance, and the RNase thus modified was fully active. Hence, 19F NMR analysis of protein, via the reaction with p-fluorobenzenesulfonyl chloride, provided not only information about the protein conformation but also direct measurements of the modification status.     

Characterization of hydrophobic cores in apomyoglobin: a proton NMR spectroscopy study

A proton nuclear magnetic resonance spectroscopic study of horse apomyoglobin was undertaken in order to define the regions of myoglobin that are and that are not structurally affected by the binding of the prosthetic group. It was found that, in spite of the poor spectral resolution, a number of spin systems could be identified by using standard correlated methods. Four clusters consisting mostly of hydrophobic residues were detected by nuclear Overhauser spectroscopy, two of which involved the tryptophan side chains. Extensive similarities to nuclear Overhauser spectroscopy data collected on the carbonmonoxy form of holomyoglobin suggested tentative assignments for several residues. It appeared that distinct cores of side chains on the distal side of the binding pocket and between the A, B, G, and H helices maintain the same packing as they do in holomyoglobin and apomyoglobin reconstituted with protoporphyrin IX.     

Sequence-specific 1H NMR assignments and secondary structure of eglin c

Sequence-specific nuclear magnetic resonance assignments were obtained for eglin c, a polypeptide inhibitor of the granulocytic proteinases elastase and cathepsin G and some other proteinases. The protein consists of a single polypeptide chain of 70 residues. All proton resonances were assigned except for some labile protons of arginine side chains. The patterns of nuclear Overhauser enhancements and coupling constants and the observation of slow hydrogen exchange were used to characterize the secondary structure of the protein. The results indicate that the solution structure of the free inhibitor is very similar to the crystal structure reported for the same protein in the complex with subtilisin Carlsberg. However, a part of the binding loop seems to have a significantly different conformation in the free protein.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations

Two different, theoretical studies of intramolecular proton-proton distances in polypeptide chains are described. Firstly, the distances between amide, C^α and C^β protons of neighbouring residues in the amino acid sequence, which correspond to the sterically allowed values for the dihedral angles φ_i, ψ_i and χ_i¹, were computed. Secondly, the frequency with which short distances occur between amide, C^α and C^β protons of neighbouring and distant residues in the amino acid sequence were statistically evaluated in a representative sample of globular protein crystal structures. Both approaches imply that semi-quantitative measurements of short, non-bonding proton-proton distances, e.g. by nuclear Overhauser experiments, should present a reliable and generally applicable method for sequential, individual resonance assignments in protein ¹H nuclear magnetic resonance spectra. Similar calculations imply that corresponding distance measurements can be used for resonance assignments in the side-chains of the aromatic amino acid residues, asparagine and glutamine, where the complete spin systems cannot usually be identified from through-bond spin-spin coupling connectivities.