In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl. This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.

The A domain of the mannitol-specific EII, IIAmtl, was subcloned and proven to be functional in the isolated form (Van Weeghel et al., 1991). It contains a histidine phosphorylation site, the first of two phosphorylation sites in the parent protein. In this paper, we describe the characterization of the three histidine residues in IIAmtl with respect to their protonation and hydrogen bonding state, using 1H[15N] heteronuclear NMR techniques and protein selectively enriched with [delta 1,epsilon 2-15N]histidine. The active site residue has a low pKa (less than 5.8) and shows no hydrogen bond interactions. The proton in the neutral ring is located at the N epsilon 2 position, which also proved to be the site of phosphorylation. The phosphorylation raises the pKa of the active site histidine considerably but does not change the hydrogen bond situation. The other two histidine residues, one of which is probably located on the surface of the protein, were also characterized. Both show hydrogen bond interactions in the unphosphorylated protein, but these are disturbed by the phosphorylation process. These observations, combined with small changes in pKa and titration behavior, indicate that the IIAmtl changes its conformation upon phosphorylation.

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The structure of the central repetitive domain of high molecular weight (HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type I1 p-turns at positions predicted from the model studies; type I1 p-turns occurred at QPGQ and QQGY sequences and type I p-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of p-turns. The type I p-turns are lost when the protein is dried on a solid surface, probably by conversion to type I1 p-turns. The homogeneous type I1 p-turn distribution is compatible with the formation of a P-spiral structure, which provides the protein with elastic properties. The p-turns and thus the P-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.Keywords: elastic properties; repetitive proteins; structural characterization; wheat gluten proteins The central repetitive domain of high molecular weight proteins forms 60-80% of their amino acid sequence and is built from the consensus peptides PGQGQQ, GYYPTS(P/L)QQ, and GQQ Shewry et al., 1994). Its structural organization may consist of a series of p-turns that organize in a @spiral structure (Tatham et al., 1984;Shewry et al., 1994); this idea is supported by scanning tunneling microscopy images of HMW Dx5 (Miles et al., 1991). The proposed &spiral structure for HMW proteins is similar to that of elastin, where it forms the basis for the molecule's elastic properties (Uny, 1993). The structural analogy between HMW proteins and elastin suggests that the HMW proteins might have elastic properties as well; however, no Reprint requests to: G.T. Robillard, Department of Biochemistry and Biophysical Chemistry and the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; e-mail: G.T.Robillard@chem.rg.nl.'Present address: Gist Brocades N.V. Postbus 1, 2600 MA Delft, The Netherlands.Abbreviurions: FITR, fourier transform infrared; HMW, high molecular weight; IPTG, isopropyl-P-D-thiogalactopyranoside. detailed structural characterization of the HMW repetitive domain has been performed. In the companion paper (van Dijk et al., 1997), we described the structure of the consensus sequences of the repetitive domain using cyclic and linear peptides. We showed that they are highly structured and contain a mixture of type I and I1 p-turns that are stabilized by mu...

Of the aromatic 'H-NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a throughspace ring-current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P-450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation.As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P-45OlI, (CYPllB1)-dependent and P-450,,, (CYP1 lA1)-dependent substrate conversions were studied. No influence on V,,, values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the K, values were increased, as was the K, value for binding of CYPl 1Al to the IH56Tladreno-doxin.The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary-structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent-exposed region of the molecule.Keyword,s. Adrenodoxin ; 'H-NMR ; aromatic region ; fluorescence.Adrenodoxin is a member of the ferredoxin family of proteins, that are widely distributed in bacteria, plants and animals. As a rule, the ferredoxins are low-molecular-mass proteins (6000-25 000 Da) that are negatively charged at neutral pH and all contain iron-sulfur clusters as the redox-active group. Bovine adrenodoxin is involved in two electron-transfer systems in the inner mitochondrial membrane of bovine adrenal cortex. Both systems contain NADPH-dependent adrenodoxin reductase, adrenodoxin and the specific cytochrome P-450, cytochrome P-450,,, (CYPllAl) or cytochrome P-45OI,,, (CYPllBl).The three-dimensional structure of adrenodoxin has not yet been elucidated and there are few data on the structural basis of the mechanism of protein-protein interaction among adrenodoxin and its redox partners. The shuttle model (Lambeth et al., 1979;Hanukoglu and Jefcoate, 1980), a ternary complex formation of adrenodoxin reductase, adrenodoxin, and the cytochrome P-450 (Kido and Kimura, 1979), and a model suggesting Correspondence to R. Bemhardt, Max-Delbriick-Centrum fur Molekulare Medizin,