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.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. II. Polarization of histidine 57 by substrate analogues and competitive inhibitors

The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin A_δ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regions. Borate ion, benzene boronic acid and 2-phenylethylboronic acid, when bound to chymotrypsin at pH 9.5 shift the resonance position of the His-Asp hydrogen bonded proton to ?15.9, ?16.3 and ?17.2 parts per million, respectively. These positions are intermediate between the low pH position in the free enzyme of ?18.0 parts per million and the high pH position of ?14.9 parts per million. The presence of these analogues prevents the His-Asp proton resonance from titrating in the region of pH 6 to 9.5. Similar low field shifts are observed for the hydrogen bonded proton resonance of subtilisin BPN′ when complexed with these boronic acids. The results support the chemical and crystallographic data which show that negatively charged tetrahedral adducts of the boronic acid substrate analogues are formed at the active sites of these enzymes. When combined with similar nuclear magnetic resonance data for the binding of N-acetyl-l-tryptophan to chymotrypsin A_δ, they suggest that a direct interaction occurs between the active site histidine and the atom occupying the leaving group position of the substrate, presumably a hydrogen bond.The His-Asp proton resonance was also monitored in complexes of chymotrypsin A_δ with bovine pancreatic trypsin inhibitor over the pH range 4 to 9. In the complex the low field proton resonance had a field position of ?14.9 parts per million over the pH range 4 to 9 indicating that His57 is in the neutral form, similar to the active enzyme at high pH.     

Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances

The use of proton-proton nuclear Overhauser enhancement (NOE) distance information for identification of polypeptide secondary structures in non-crystalline proteins was investigated by stereochemical studies of standard secondary structures and by statistical analyses of the secondary structures in the crystal conformations of a group of globular proteins. Both regular helix and beta-sheet secondary structures were found to contain a dense network of short 1H-1H distances. The results obtained imply that the combined information on all these distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra is sufficient for determination of the helical and beta-sheet secondary structures in small globular proteins. Furthermore, cis peptide bonds can be identified from unique, short sequential proton-proton distances. Limitations of this empirical approach are that the exact start or end of a helix may be difficult to define when the adjoining residues form a tight turn, and that unambiguous identification of tight turns can usually be obtained only in the hairpins of antiparallel beta-structures. The short distances between protons in pentapeptide segments of the different secondary structures have been tabulated to provide a generally applicable guide for the analysis of NOESY spectra of proteins.     

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.     

Protein dynamics by solid-state nuclear magnetic resonance spectroscopy: Peptide backbone of the coat protein in fd bacteriophage

¹³C and ¹⁵N chemical shift anisotropy and ¹⁵N¹H dipolar powder patterns from backbone sites of the coat protein in fd bacteriophage are not averaged by motion. This means that the polypeptide backbone of the protein has no large amplitude motions rapid compared to 10⁴ Hz. Relaxation studies on the ¹³C_α and ¹⁵N amide resonances indicate the presence of motions on the 10⁹ Hz timescale. These results are reconciled with a model where an otherwise rigid backbone undergoes small amplitude, rapid motions.     

Repressor-operator interaction in the lac operon: III. Nuclear magnetic resonance observations with altered amino-terminal DNA binding domains

We have examined the N-terminal 56 amino acid fragment, the domain that can bind DNA independently, from 3-fluorotyrosine-substituted Escherichia coli lac repressor by ¹⁹F-nuclear magnetic resonance. The fragments or “headpieces” from four altered repressers missing each of the tyrosines in turn were examined in parallel. When the wild-type N-terminal fragment is titrated with a 36 base-pair lac operator DNA sequence, the ¹⁹F resonances undergo changes in their chemical shifts that are different from those changes when the N-terminal fragment is titrated with non-specific DNA fragments. By looking at these operator-induced changes as well as pH-dependent effects with all four altered N-terminal fragments, we show systematic correlations with the genetic data. The data lead us to conclude that upon operator DNA binding: (1) tyrosine 7 is displaced to a less polar environment and the higher than normal pK value of the phenolic OH group is decreased; (2) tyrosine 12 does not change much in either its mobility or environment; and (3) tyrosine 17 is involved, as suggested by the genetic data, when the headpiece forms a complex with operator DNA.     

Calibration of the angular dependence of the amide proton-C alpha proton coupling constants, 3JHN alpha, in a globular protein. Use of 3JHN alpha for identification of helical secondary structure

The vicinal amide proton-C alpha proton spin-spin coupling constants, JHN alpha, in the globular protein basic pancreatic trypsin inhibitor (BPTI) have been measured using phase-sensitive correlated spectroscopy at high digital resolution. In conjunction with the crystal structure of BPTI, these data were used to calibrate the correlation between 3JHN alpha and the dihedral angle phi. The resulting "BPTI curve" is 3JHN alpha = 6.4 cos2 theta - 1.4 cos theta + 1.9 (theta = [phi - 60 degrees]). It is further shown that measurement of the spin-spin couplings 3JHN alpha presents an independent, reliable method for identification of the location of helical structure in the amino acid sequence of proteins.     

A protein structure from nuclear magnetic resonance data. lac repressor headpiece

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.     

Model-building of neurohypophyseal hormones

An attempt is made to construct models of the polypeptide backbone of the neurohypophyseal hormones. Using the sequences of six hormones and the empirical statistical approach (Kabat &; Wu, 1972; Wu &; Kabat, 1973), an initial selection is made of a set of φ, ψ angles satisfying all six sequences from a 20 × 20 × 20 table of φ, ψ values of tripeptides in 11 known proteins. Because of insufficient data at several positions, φ, ψ angles in the α-helical and β-sheet domains were considered as possible alternatives and the final selection was made to bring the disulfide bond as close as possible (model I). These initial values are then subjected to an energy minimization to refine the model; reasonable agreement with nuclear magnetic resonance data for possible φ values is obtained. Model II is constructed to be consistent with the available nuclear magnetic resonance data and the suggestion of a sequential Cu²⁺-binding site. Both models contain similar structural features and may be converted to one another with rotation of only a few angles. Interactive computer graphics was applied to construct model II, and its use in problems in this type is described. The relation of the experimental data to the various models that have been proposed for the neurohypophyseal hormones is discussed and an experiment is suggested that may permit a choice between them.     

Molecular conformation of alpha-bungarotoxin as studied by nuclear magnetic resonance and circular dichroism

We have examined the circular dichroism and nuclear magnetic resonance spectra of a long neurotoxin, alpha-bungarotoxin, over a wide range of pH values and temperatures, and under high salt conditions. The observations are interpreted partly in terms of the known crystal structure of this polypeptide. We support earlier findings of a greater degree of beta-sheet structure in solution than has been reported by X-ray crystallography and, importantly, the invariant residue associated with neurotoxicity, Trp29, is shown to be in a similar environment to that found in alpha-cobratoxin and LS III from Laticauda semifasciata. The implications of this observation for structure/function relationships are outlined.     

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.     

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Effects of N-terminal extension peptides on the structure and stability of bovine pancreatic trypsin inhibitor studied by H n.m.r.

Four N-terminal extended species of the wild-type bovine pancreatic trypsin inhibitor (WT-BPTI), Arg-BPTI (1-BPTI), Met-Glu-Ala-Glu-BPTI (4-BPTI), Ser-Ile-Glu-Gly-Arg-BPTI (5-BPTI) and Gly-Ser-Ile-Glu-Gly-Arg-BPTI (6-BPTI) have been studied by 1H n.m.r. The overall structure of the protein is largely unaffected by the addition of extension peptides. pH titration effects on the C-terminal Ala 58 H beta chemical shift indicate that the structure of 1-BPTI at neutral pH is very similar to that of the WT protein, with a salt bridge between the main chain terminal charges. A salt bridge interaction is prevented by addition of the longer extension peptides. Temperature stabilities are measured by high temperature hydrogen isotope exchange and by microcalorimetry. The stability of 1-BPTI is equal to that of WT-BPTI. A slight decrease in stability is observed for longer extensions, following the order WT-BPTI = 1-BPTI < 5-BPTI = 6-BPTI < 4-BPTI. Small changes in chemical shift are observed for 30 invariant resonances in 4-, 5- and 6-BPTI and for a subset of this group in 1-BPTI. These protons are distributed over about half of the BPTI molecule. The size of the chemical shift changes for many resonances follow the same ranking as the temperature stability. The chemical shift effects are attributed to charge and dielectric effects from extension peptides that probably share a common orientation on the surface of BPTI.     

Protein folding: evaluation of some simple rules for the assembly of helices into tertiary structures with myoglobin as an example

A computer program designed to fold a peptide chain consisting solely of helical segments and connecting links of known length is described and evaluated. This study is a detailed extension of certain aspects of the earlier work of Ptitsyn &; Rashin (1975). Possible interaction sites on the helices are sequence dependent and are calculated as described by Richmond &; Richards (1978) using probable changes in solvent contact area as a guide. The helices are then paired according to the list of potential sites, with each helix being paired at least once. The lists of pairings are then examined geometrically, each site having a defined dihedral helix axis angle, a specified inter-helix axis distance, and defined rotations, when required, about each helix axis. Two simplified filters are used: (1) lengths of connecting links must be equal to or greater than the end-to-end distances of the helices; and (2) non-paired helices must not collide. With myoglobin as a test example and only six of the eight helices being considered, a conformation space consisting of more than 3 × 10⁸ structures was surveyed. The two filters reduced the acceptable structure list to 121. Slight readjustment of the parameters in the filters would have reduced this to 20 structures. Of these 20, one closely resembles the actual distribution of helices in myoglobin. The possible utility and pitfalls of this approach as part of an overall protein folding program are discussed.     

Quantitative determination of conformations of flexible molecules in solution using lanthanide ions as nuclear magnetic resonance probes: application to adenosine-5'-monophosphate

A procedure is described here whereby the conformation, of a flexible molecule in solution can be found. The method depends on the study of the nuclear magnetic resonance spectrum of the molecule in the presence of perturbations due to specifically bound lanthanide cations. The magnetic perturbations are of two kinds: shifts of nuclear magnetic resonance spectral lines in the presence of cations such as Eu³⁺ and changes in relaxation rates of the nuclear magnetic resonance excitations in the presence of cations such as Gd³⁺. Suitable expressions are given for the relation between the magnitude of the perturbations and the geometry of the lanthanide complex in the absence of through-bond perturbations and for an axially symmetric system. It is proved that the spectral changes described here are not due to through-bond (contact) effects. The circumstances, in which the anisotropy of the magnetic susceptibility tensor, as seen in the nuclear magnetic resonance spectra, is of axial symmetry, are defined. The experimental systems described are of this kind. A computer program has been devised that searches for the conformations of the molecule which fit the nuclear magnetic resonance data.We outline here the principles of the method and how we have used a combination of relaxation and shift probes to obtain the conformation of adenosine-5′-monophosphate at pH 2. It is shown that a small family of closely related conformations fit the nuclear magnetic resonance data. These conformations are very similar to that of the crystal structure of AMP.     

Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution

The X-ray structure of a new crystal form of chymotrypsinogen A grown from ethanol/water has been determined at 1.8 A resolution using Patterson search techniques. The crystals are of orthorhombic space group P212121 and contain two molecules in the asymmetric unit. Both independent molecules (referred to as A and B) have been crystallographically refined to a final R value of 0.173 with reflection data to 1.8 A resolution. Owing to different crystal contacts, both independent molecules show at various sites conformational differences, especially in segments 33-38, 142-153 and 215-222. If these three loops are omitted in a comparison, the root-mean-square (r.m.s.) deviation of the main-chain atoms of molecules A and B is 0.32 A. If segments 70-79, 143-152 and 215-221 are omitted, a comparison of either molecule A or molecule B with the chymotrypsinogen model of Freer et al. (1970) reveals an r.m.s. deviation of the alpha-carbon atoms of about 0.7 A. Compared with the active enzyme, four spatially adjacent peptide segments, in particular, are differently organized in the zymogen: the amino-terminal segment 11-19 runs in a rigid but strained conformation along the molecular surface due to the covalent linkage through Cys1; also segment 184-194 is in a rigid unique conformation due to several mutually stabilizing interactions with the amino-terminal segment; segment 216-222, which also lines the specificity pocket, adapts to different crystal contacts and exists in both chymotrypsinogen molecules in different, but defined conformations; in particular, disulfide bridge 191-220, which covalently links both latter segments, has opposite handedness in molecules A and B; finally, the autolysis loop 142 to 153 is organized in a variety of ways and in its terminal part is completely disordered. Thus, the allosteric activation domain (Huber & Bode, 1978) is organized in defined although different conformations in chymotrypsinogen molecules A and B, in contrast to trypsinogen, where all four homologous segments of the activation domain are disordered. This reflects the structural variability and deformability of the activation domain in serine proteinase proenzymes. If the aforementioned peptide segments are omitted, a comparison of our chymotrypsinogen models with gamma-chymotrypsin (Cohen et al., 1981) yields an r.m.s. deviation for alpha-carbon atoms of about 0.5 A. The residues of the "active site triad" are arranged similarly, but the oxyanion hole is lacking in chymotrypsinogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rigidification of a flexible protease inhibitor variant upon binding to trypsin

The Tyr35-->Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 A data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.