Herrn Professor Rolf Huisgen zum 85. Geburtstag gewidmetThe NMR-solution structure of an a-heptapeptide with a central Aib residue was investigated in order to verify that, in contrast to b-peptides, short a-peptides do not form a helical structures in MeOH. Although the central Aib residue was found to induce a bend in the experimentally determined structure, no secondary structure typical for longer a-peptides or proteins was found. A b 2 /b 3 -nonapeptide with polar, positively charged side chains was subjected to NMR analysis in MeOH and H 2 O. Whereas, in MeOH, it folds into a 10/12-helix very similar to the structure determined for a corresponding b 2 /b 3 -nonapeptide with only aliphatic side chains, no dominant conformation could be determined in H 2 O. Finally, the NMR analysis of a b 3 -icosapeptide containing the side chains of all 20 proteinogenic amino acids in MeOH is described. It revealed that this 20mer folds into a 3 14 -helix over its whole length forming six full turns, the longest 3 14 -helix found so far. Together, our findings confirm that, in contrast to a-peptides, b-peptides not only form helices with just six residues, but also form helices that are longer than helical sections usually observed in proteins or natural peptides. The higher helix-forming propensity of long b-peptides is attributed to the conformation-stabilizing effect of the staggered ethane sections in b-peptides which outweighs the detrimental effect of the increasing macrodipole.

In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole-dipole interactions and the associated ''spin diffusion'' due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment.

The structural properties of an all-b 3 -dodecapeptide with the sequence H-b-HLys(N e -CO(CH 2 ) 3 -SÀAcm)-b-HPhe-b-HTyr-b-HLeu-b-HLys-b-HSer-b-HLys-b-HPhe-b-HSer-b-HVal-b-HLys-b-HAla-OH (1) have been studied by two-dimensional homonuclear 1 H-NMR and by CD spectroscopy. In MeOH solution, highresolution NMR spectroscopy showed that the b-dodecapeptide forms an (M)-3 14 -helix, and the CD spectrum corresponds to the pattern expected for an (M)-3 14 -helical secondary structure. In aqueous solution, however, the peptide adopts a predominantly extended conformation without regular secondary-structure elements, which is in agreement with the absence of the characteristic trough near 215 nm in the CD spectrum. The NMR and CD measurements with solutions of 1 in MeOH containing 3m urea further indicated that the peptide retains the regular secondary structural elements under these conditions, whereas, after addition of 40% (v/v) H 2 O to the MeOH solution, the large 1 H-chemical-shift dispersion indicative of a defined spatial peptide fold was lost. The b 3 -dodecapeptide is ± so far ± the longest b-peptide shown to adopt a regular (M)-3 14 -helix conformation in an organic solvent. The observation that the structure of this long b 3 -peptide is not maintained in aqueous solution indicates that the (M)-3 14 -fold is primarily stabilized by short-range interactions.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of the intact enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3'-hydroxyl group of UDP-N-acetylglucosamine (UDPNAG). This reaction constitutes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan (murein). The transfer reaction involves the nucleophilic attack of the 3'-hydroxyl group of UDPNAG at the C-2 of PEP. The three-dimensional structure of MurA complexed with UDPNAG revealed an aspartate residue (D305 in the En. cloacae sequence) close to the 3'-hydroxyl group of UDPNAG, suggesting that it may act as an acid-base catalyst in the active center of the enzyme. In addition to aspartate 305, asparagine 23 also interacts with the 3'-hydroxyl group; however, its role in catalysis or binding of the UDPNAG substrate is unclear. To gain information on the role of these two amino acids in the MurA-catalyzed reaction we have exchanged D305 for alanine, cysteine, histidine, and glutamate, and N23 for alanine and serine using site-directed mutagenesis. While the D305 alanine, cysteine, and histidine mutant proteins do not have detectable enzymatic activity, the D305E mutant protein exhibits a low residual activity (ca. 0.1% of the wild-type enzyme). Unlike with wild-type MurA, no exothermic signal was obtained when the D305A and -E mutant proteins were titrated with UDPNAG, demonstrating that the affinity of the sugar nucleotide substrate is reduced as a result of the amino acid exchange. The reduced affinity to UDPNAG leads to a lower propensity of C115 to form either the O-phosphothioketal with PEP or the thioether with the antibiotic fosfomycin. These findings emphasize the dual role of D305 as a general base and an essential binding partner to UDPNAG in the active site of MurA. Similarly, the two N23 mutant proteins showed a much lower catalytic activity although binding of UDPNAG was not as much affected as in the case of the D305 mutant proteins. This result indicates that this amino acid residue is mainly involved in stabilization of transition states.

Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form.

This paper describes the NMR screening of 141 small (<15 kDa) recombinant Thermotoga maritima proteins for globular folding. The experimental data shows that approximately 25% of the screened proteins are folded under our screening conditions, which makes this procedure an important step for selecting those proteins that are suitable for structure determination. A comparison of screening based either on 1D 1H NMR with unlabeled proteins or on 2D [1H,15N]-COSY with uniformly 15N-labeled proteins is presented, and a comprehensive analysis of the 1D 1H NMR screening data is described. As an illustration of the utility of these methods to structural proteomics, the NMR structure determination of TM1492 (ribosomal protein L29) is presented. This 66-residue protein consists of a N-terminal 3(10)-helix and two long alpha-helices connected by a tight turn centered about glycine 35, where conserved leucine and isoleucine residues in the two alpha-helices form a small hydrophobic core.

SummaryCell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear 1 H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-a-helix bundle with an up-down-up topology and a 3 10 -helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters. IUBMB Life, 59: 578-585, 2007

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