NMR investigation and secondary structure of domains I and II of rat brain calbindin D28k (1-93).

Calbindin D28k, a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1-93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1H, 13C and 15N resonances. The secondary structure of the protein was deduced from the size of the 3JHN-Halpha coupling constants, the chemical shift indices of 1Etaalpha, 13Calpha, 13C' and 13Cbeta nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major alpha-helices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76-Leu90, while residues Ala2-Leu6 form a fifth, flexible helical segment. Two short beta-strands (Tyr30-Glu32, Lys72-Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

The three-dimensional high resolution structure of human interferon alpha-2a determined by heteronuclear NMR spectroscopy in solution.

The solution structure of recombinant human interferon alpha-2a (Roferon-A) has been determined by multidimensional heteronuclear NMR spectroscopy. The calculations using simulated annealing produced a family of 24 convergent structures which satisfy the experimental restraints comprising 1541 NOE-derived inter-proton distances, 187 dihedral restraints, 66 pairs of hydrogen bond restraints, and six upper and lower limits for two disulfide bridges. The fractional labeling of methyl groups allowed their direct and unambiguous stereospecific assignment which proved to be essential for obtaining a high resolution of the structures. A best fit superposition of residues 10 to 47, 50 to 101 and 111 to 157 gives an rms deviation of 0.62 A for the backbone heavy atoms and 1.39 A for all heavy atoms of these segments. The dominant feature of the structure is a cluster of five alpha-helices, four of which are arranged to form a left-handed helix bundle with an up-up-down-down topology and two over-hand connections. The interpretation of heteronuclear 15N-¿1H¿ NOE data shows the co-existence of flexible regions within an otherwise rigid framework of the protein. Four stretches of pronounced flexibility can be located: Cys1-Ser8, Gly44-Ala50, Ile100-Lys112, and Ser160-Glu165. Among the structurally related four-helical bundle cytokines, the structure of IFN alpha-2a is most similar to that of human interferon alpha-2b and murine interferon-beta. From this structural information and mutagenesis data, areas on the surface of the protein are identified which seem to be important in receptor interactions.

NMR studies of DNA duplexes singly cross-linked by different synthetic linkers.

Molecular modelling studies resulted in the design of a variety of non-nucleotidic covalent linkers to bridge the 3'-end of the (+)-strand and the 5'-end of the (-)-strand in DNA duplexes. Three of these linkers were synthesized and used to prepare singly cross-linked duplexes d(GTGGAATTC)-linker-d(GAATTCCAC). Linker I is an assembly of a propylene-, a phosphate- and a second propylene-group and is thought to mimic the backbone of two nucleotides. Linkers II and III consist of five and six ethyleneglycol units, respectively. The melting temperatures of the cross-linked duplexes are 65 degrees C for I and 73 degrees C for II and III, as compared with 36 degrees C for the corresponding non-linked nonadeoxynucleotide duplex. The three cross-linked duplexes were structurally characterized by nuclear magnetic resonance spectroscopy. The 1H and 31P resonance assignments in the DNA stem were obtained using standard methods. For the resonance assignment of the linker protons, two-dimensional 1H-31P heteronuclear COSY and two-quantum-experiments were used. Distance geometry calculations with NOE-derived distance constraints were performed and the resulting structures were energy-minimized. In duplex I, the nucleotides flanking the propylene-phosphate-propylene-linker do not form a Watson-Crick base pair, whereas in duplexes II and III the entire DNA stem is in a B-type double helix conformation.

Direct NMR evidence for an intermediate preceding the rate-limiting step in the unfolding of ribonuclease A.

It is commonly believed that there are no detectable intermediates in the kinetic unfolding reactions of small proteins. If such intermediates could be found, they would give important information about the nature of the transition state for unfolding, which is thought to occur close to the native state. We report here that one-dimensional proton magnetic resonance spectra recorded during the unfolding of ribonuclease A provide direct evidence for at least one unfolding intermediate in which side chains are free to rotate. This intermediate appears to be a 'dry molten globule' of the kind hypothesized by Shakhnovich and Finkelstein.

1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-gamma.

1H, 13C, and 15N NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz 13C alpha and 1H alpha line widths of this high molecular weight dimer and the extensive overlap in the 1H alpha and 13C alpha spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the 15N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the 15N-separated 3D NOESY-HMQC and 13C/15N-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C alpha and C beta chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-702], i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.

Selective interaction of Ni with an MHC-bound peptide.

T cells generally recognize foreign antigens as peptides associated with self-molecules encoded by genes of the major histocompatibility complex (MHC). However, T cells which are specific for non-peptidic haptens have been described, in particular in patients with contact sensitivity reactions to metals such as nickel (Ni). Previously, we isolated MHC class II-restricted Ni-specific T cell clones from patients with Ni allergy. The experiments reported here examine the molecular basis for the interaction between Ni and peptide-MHC complexes. We find that Ni alters a T cell response to a peptide and show that Ni interacts with this peptide to alter its antigenicity rather than its ability to bind to MHC molecules. These findings hold implications for a model of hapten recognition by T cells.

Anantin--a peptide antagonist of the atrial natriuretic factor (ANF). II. Determination of the primary sequence by NMR on the basis of proton assignments.

Anantin, a naturally occurring peptide from Streptomyces coerulescens, binds competitively to the receptor of atrial natriuretic factor (ANF) from bovine adrenal cortex (Kd = 0.6 microM) and acts as ANF antagonist. Protein chemical data and FAB-MS have identified anantin to be a cyclic polypeptide consisting of 17 common L-amino acids. The molecule is highly stable and precludes the application of standard sequencing methods. The primary sequence of anantin was determined by 2D 1H NMR spectroscopy and the application of advanced protein chemical methods to be Gly1-Phe2-Ile3-Gly4-Trp5-Gly6-Asn7-Asp8 -Ile9-Phe10-Gly11-His12-Tyr13-Ser14+ ++- Gly15-Asp16-Phe17. The molecule is cyclized between the beta-carboxyl group of Asp8 and the amino group of Gly1.

Secondary structure determination for alpha-neurotoxin from Dendroaspis polylepis polylepis based on sequence-specific 1H-nuclear-magnetic-resonance assignments.

Sequence-specific assignments are presented for the polypeptide backbone protons and a majority of the amino-acid-side-chain protons of alpha-neurotoxin from Dendroaspis polylepis polylepis, and individual amide proton-exchange rates with the solvent are reported. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The molecule includes a three-stranded antiparallel beta-sheet, and there are indications that two additional short chain segments are arranged in an antiparallel beta-sheet. These structural elements are similar, but not identical, to either the secondary structure reported for erabutoxin b in single crystals, or the solution structure of cytotoxin CTXIIb from Naja mossambica mossambica.

Kinetic circular dichroism shows that the S-peptide alpha-helix of ribonuclease S unfolds fast and refolds slowly.

It is shown that circular dichroism (CD) can distinguish between the S-peptide and the S-protein fragments of RNase S at 225 nm and 235 nm. The conformational source for the strong CD at 225 nm is the S-peptide alpha-helix. The structural assignment of the CD at 235 nm is not clear but it is shown to be largely due to the S-protein moiety. This situation is utilized to monitor the kinetics of pH-induced unfolding and refolding of the two moieties. It is observed that major changes occur both in the fast and slow phases of unfolding as well as refolding. Specifically, the S-peptide alpha-helix unzippering is a fast reaction, followed by slow kinetics only at 235 nm. These latter kinetics parallel the appearance of the slow-folding species commonly attributed to the accumulation of non-native proline isomers. In refolding, a large fraction of the CD of S-protein at 235 nm recovers rapidly. The S-peptide alpha-helix zippers up last. These results are unexpected and their implications for the folding mechanism of ribonuclease are discussed.

Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions.

The kinetics of the Quin 2-Ca2+ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca2+ complex with a large excess of EGTA, EDTA or MgCl2 resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83 +/- 4 s-1 for the dissociation rate constant (k-) at pH 7.2, 37 degrees C, +/- 3mM Mg2+. The temperature dependence of the dissociation was weak (activation energy = 22 +/- 1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k+ was estimated to be greater than or equal to 10(9) M-1s-1, in agreement with the calculation from k+ = k-/K. These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca2+ transients.

Measurement of the refolding combination reaction between S-peptide and S-protein.

S-Peptide combines with S-protein during the refolding of ribonuclease S. The kinetics of combination have now been measured by a specific probe, the absorbance (492 nm) of a fluoresceinthiocarbamyl (FTC) group on lysine-7 of S-peptide. pK changes of the FTC group detect both initial combination and later, first-order, stages in folding. Combination with the slow-folding species of S-protein occurs with a half-time of 0.4 s at 50 microM, whereas complete folding takes 50 s (pH 6.8, 31 degrees C). Thus combination takes place at an early stage in folding. The second-order rate constant of the refolding combination reaction (5 X 10(4) M-1 s-1) is 100-fold smaller than that for combination with folded S-protein, which probably reflects the lower affinity of S-protein for S-peptide in the initial complex. Inhibition by S-peptide of combination between FTC-S-peptide and S-protein shows that the refolding combination reaction is specific and reversible. Both the fast-folding and slow-folding species of unfolded S-protein participate in the refolding combination reaction.

Nature of the fast and slow refolding reactions of iron(III) cytochrome c.

The fast and slow refolding reactions of iron(III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. (Ikai, A., Fish, W. W., & Tanford, C. (1973) J. Mol. Biol. 73, 165--184), have been reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25 degrees C, 1.75 M guanidine hydrochloride). We show here that native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes have been used to test for formation of native cyt c. absorbance in the 695-nm band and rate of reduction of by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown both by refolding assays at different times after unfolding ("double-jump" experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF leads to N and the slow refolding reaction is Us leads to N, where N is native cyt c, and there is a US in equilibrium UF equilibrium in unfolded cyt c. The results are consistent with the UF in equilibrium US reaction being proline isomerization, but this has not yet been tested in detail. Folding intermediates have been detected in both reactions. In the UF leads to N reaction, the Soret absorbance change precedes the recovery of the native 695-nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US leads to N reaction an ascorbate-reducible intermediate has been found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.