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and cross peaks corresponding to H1 and another proton (H3) that is scalar coupled to H2. This procedure is continued by selecting the protons that are scalar coupled to H3 from the plane (C3) that displays the proper pattern of proton signals (diagonal peak at H3, cross peak at H2). Ambiguities in the assignments which arise from protons resonating at the same frequency can be resolved in this experiment by editing the COSY data by the I3C frequencies. For example, although the 1 and 3 protons of ring A have nearly identical chemical shifts, the shifts of the attached carbons (C1 and C3) are different. Thus, as shown in column 1 of Figure 2, the COSY responses involving the degenerate signals of H1 and H3 can be distinguished by their location in different planes (Cl , H6-Hl-H2; C3,H4-H3-H2). Analogous to a two-dimensional heteronuclear relay experiment,I0 the 3D HMQC-COSY experiment provides relay information by correlating the chemical shifts of a carbon, the attached proton(s), and their scalar coupled partners. In contrast to the heteronuclear relay experiment, however, the 3D experiment uniquely defines the frequency of the relay spin.'s3 This is important in cases where I3C signals overlap such as C3 and C4 in ring B of kanamycin A. Since the relay spins (H3, H4) are uniquely defined in w2 in the 3D experiment, the H2-H3-H4 and H3-H4-H5 connectivity patterns can easily be distinguished (Figure 2). In summary, heteronuclear 3D N M R spectroscopy is a useful method for resolving spectral overlap in all frequency domains. This is important for assigning spectra and elucidating the structures of complicated molecules. In addition, the increase in resolution afforded by this technique will help to automate peak-picking and assignment procedures and facilitate the extraction of J couplings (HMQC-COSY) and quantitative NOE information (HMQC-NOESY) used in the determination of three-dimensional structures.

Peroxidation of SCN − to OSCN − , catalysed by myeloperoxidase and lactoperoxidase, was studied. The rate of this reaction showed sharp optima between pH 5 and 7.5, the position of which is determined by the concentrations of both SCN − and H 2 O 2 . At low pH values, both SCN − and H + inhibited myeloperoxidase and lactoperoxidase competitively with respect to H 2 O 2 . The inhibition constants of SCN − for myeloperoxidase and lactoperoxidase (2 and 6 mM, respectively) are independent of pH. For these enzymes a K i for H + of 1 μM was found that corresponded to an ionisable group on the enzymes (p K a = 6) which controls the enzymic activity. A kinetic expression is proposed that explains most of the data. The physiological consequences of the corresponding mechanism are discussed.

Repressors are proteins that regulate transcription by binding to DNA control regions (the so-called operators). The classic system for regulation of gene expression is the lac Operon of E. coli. Many ideas on negative control of transcription, and, indeed, the now familiar concept of an operon, were originally derived from a study of the E. coli lactose genes (Jacob and Monod, 1961). Lac repressor was also the first repressor that was isolated (Gilbert and Muller-Hill, 1966). Over the years, it has been the subject of numerous biochemical and genetic studies (for reviews see Bourgeois and Pfahl, 1976; Miller and Reznikoff, 1978; Miller, 1979; 1984). Initially, the lac repressor-lac operator system served as the principal model system for studies on specific DNA recognition by proteins. When it appeared that lac repressor refused to crystallize in a form suitable for X-ray crystallography, it lost its primacy and attention was turned to other proteins involved in gene regulation such as CAP and the phage λ repressors, cI and cro. The crystal structures of these proteins were solved in the early 1980s (McKay and Steitz, 1981; Pabo and Lewis, 1982; Anderson et al., 1981). This crystallographic work and its implications for protein-DNA recognition have been reviewed by Pabo and Sauer (1984). Later, the structure of the trp repressor was solved (Schevitz et al., 1985) and, at low resolution, that of a complex of 434 repressor and its cognate operator (Anderson et al., 1987).

One-dimensional and two-dimensional photochemically induced dynamic nuclear polarisation (photo-CIDNP) nuclear magnetic resonance spectra have been recorded for the sea-anemone polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxins I and II. In anthopleurin-A and toxin II, all three Trp residues are accessible to the flavin dye, although Trp-23 in anthopleurin-A shows a weaker photo-CIDNP response than Trp-33 and Trp-45. Tyr-25 in anthopleurin-A also shows a strong response. In toxin I, Trp-23, Trp-33 and Tyr-45 (which replaces Trp in this molecule) are accessible to the dye. The pH dependences of the photo-CIDNP spectra of all three polypeptides have been examined. The response of Trp-33 increases significantly with pH. The two His residues of anthopleurin-A and toxin II display a response in their imidazole forms, but not their imidazolium forms. The surface accessibilities of Trp-23 and Trp-33 are discussed in relation to the interaction of these polypeptides with the Na+ channel.

(1) In the pH range between 5.0 and 8.0, the rate constants for the reaction of ferrocytochrome c with both the high- and low-affinity sites on the cytochrome aa3 increased by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa3 did nt cause a change in the pH of an unbuffered medium. Furthermore, it was found that this reaction and the steady-state reaction are equally fast in H2O and 2H2O. From these results it was concluded that no protons are directly involved in a rate-determining reaction step. (3) Arrhenius plots show that the reaction between ferrocytochrome c and cytochrome aa3 requires a higher enthalpy of activation at temperatures below 20 degrees C (15--16 kcal/mol) as compared to that at higher temperature (9 kcal/mol). We found no effect of ionic strength on the activation enthalpy of the pre-steady-state reaction, nor on that of the steady-state reaction. This suggests that ionic strength does not change the character of these reactions, but merely affects the electrostatic interaction between both cytochromes.

This paper describes the possibility of using 2D-NMR spectroscopy in the study of the morphology of micelles. In aqueous solution, the monomeric surfactant 1-methyl-4-dodecylpyridinium iodide exhibits positive NOE's whereas negative NOE's are observed for the micellar aggregate. 1-Methyl-4-n-butylpyridinium iodide , which does not form micelles, only shows positive NOE's. It is argued that the observation of negative NOE's for micelles is reconcilable with quantitative data for the dynamics of these surfactant aggregates.

Lac repressor of E. coli recognizes a 20 to 25 basepair DNA sequence called the lac operator.1–4 When it binds to this region it blocks transcription of the genes coding for the lactose enzymes. Genetic and biochemical data have implicated the N-terminal part or ‘headpiece’ of the repressor as the DNA binding domain.4, 5 The precise way in which lac repressor recognizes its operator is, however, not known.

How do proteins such as repressors recognize specific base pair sequences in DNA? This is one of the most intriguing problems in molecular biology. Some clues have come from the well-known crystallographic studies on cro (Anderson et al. 1981) and lambda repressors (Pabo and Lewis 1982), CAP (McKay and Steitz 1981) and, more recently, trp repressor (Schevitz et al. 1985). All these proteins have a very similar helix-turn-helix structural domain, which is implicated from genetic studies to contain the main determinants for specific DNA recognition (for a review see Pabo and Sauer 1984). Solution of the repressor crystal structures led to a series of model-building studies aimed at modelling the interaction with the DNA binding sites (Anderson et al. 1981; Ohlendorf et al. 1982; Pabo and Lewis 1982; McKay and Steitz 1981; Matthews et al. 1982; Weber et al. 1982).

An experimental approach for the editing of exchange-relayed NOEs in water-selective NOE experiments is presented. The proposed pulse sequence is based on the application during the NOE mixing time of continuous wave irradiation, which saturates resonances of relaying labile protons in slow chemical exchange with water. The technique can efficiently reduce the contributions of exchange-relayed NOE peaks that often crowd the water-selective NOE spectra and hide direct intermolecular NOEs between water and protein protons. The present approach opens new opportunities for the characterization of hydration by NMR, even in the proximity of polar labile groups.

Bacteria require three initiation factors, IF1, IF2 and IF3, to start protein synthesis. In the last few years the elucidation of both structural and mechanistic aspects pertaining to these proteins has made substantial progress. In this article we outline the translation initiation process in bacteria and review these recent developments giving a summary of the main features of the structure and function of the initiation factors.

A method is proposed for the structure determination of biomolecules in solution based on 2D NOE data in which indirect magnetization transfer (spin diffusion) is taken into account. In this method experimental NOE's are supplemented with calculated NOE's based on a molecular model. Back transformation of this mixed NOE matrix gives a relaxation matrix which provides a better estimation for the cross-relaxation rates than can be obtained directly from the NOE cross-peaks. From the cross-relaxation rates distance constraints can be derived, which can be used in distance geometry and restrained molecular dynamics calculations to obtain an improved molecular model. The procedure can be repeated until all experimental NOE's are satisfactorily explained.

Background: Research on high-alkaline proteases, such as serine protease PB92, has been largely inspired by their industrial application as protein-degrading components of washing powders. Serine protease PB92 is a member of the subtilase family of enzymes, which has been extensively studied. These studies have included exhaustive protein engineering investigations and X-ray crystallography, in order to provide insight into the mechanism and specificity of enzyme catalysis. Distortions have been observed in the substrate-binding region of subtilisin crystal structures, due to crystal contacts. In addition, the structural variability in the substrate-binding region of subtilisins is often attributed to flexibility. It was hoped that the solution structure of this enzyme would provide further details about the conformation of this key region and give new insights into the functional properties of these enzymes. Results: The three-dimensional solution structure of the 269-residue (27 kDa) serine protease PB92 has been determined using distance and dihedral angle constraints derived from triple-resonance NMR data. The solution structure is represented by a family of 18 conformers which overlay onto the average structure with backbone and all-heavy-atom root mean square deviations (for the main body of the molecule) of 0.88 and 1.21 A, respectively. The family of structures contains a number of regions of relatively high conformational heterogeneity, including various segments that are involved in the formation of the substrate-binding site. The presence of flexibility within these segments has been established from NMR relaxation parameters and measurements of amide proton exchange rates. Conclusions: The solution structure of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites. Among the limited number of residues involved in significant internal mobility are those of two pockets, termed S1 and S4, within the substrate-binding site. The presence of flexibility within the binding site supports the proposed induced fit mechanism of substrate binding.

On montre que les spectroscopies CIDNP-COSY et CIDNP-NOESY sont utiles pour etudier des residus de surface et leur environnement immediat dans des macromolecules biologiques