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The conformational changes of free, monomeric glucagon-like peptide-1-(7–36)-amide (GLP-1) in aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from 292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil (in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more stable than the N-terminal part. Copyright © 2001 John Wiley & Sons, Ltd.

A new experiment for selective determination of the relaxation rates of fast relaxing NMR signals is presented. The experiment is derived from the conventional inversion recovery experiment by substituting the 180° inversion pulse of this experiment with a signal eliminating relaxation filter (SERF) consisting of three 180° pulses separated by two variable delays, Δ1and Δ2. The SERF experiment allows a selective suppression of signals with relaxation rates below a given limit while monitoring the relaxation of faster relaxing signals. The experiment was tested on a sample of 20% oxidized plastocyanin from Anabaena variabilis, where the fast exchange of an electron between the reduced (diamagnetic) and the oxidized (paramagnetic) form results in a series of average signals with widely different relaxation rates. To ensure an optimum extraction of information from the experimental data, the relaxation rates were obtained from the SERF experiment by a simultaneous analysis of all the FIDs of the experiment using a fast linear prediction model method developed previously. The reliability of the relaxation rates obtained from the SERF experiment was confirmed by a comparison of the rates with the corresponding rates obtained from a conventional inversion recovery experiment.

The human a3-chain type VI collagen C-terminal Kunitz domain fragment (a3(VI)) has been studied by two-dimensional 1H-1H and 1H-13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual Ha chemical shifts, which are highly conserved in Kunitz-domain proteins, are also observed for a3(VI). Further-more a series of exchange cross peaks observed in 1H-1H spectra shows that a large number of protons in the central ß-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser47(53) Ha, Arg32(38) H?2, and Gln48(54) Hß2, all located in the vicinity of the Trp21(27) ring in the crystal structure of a3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609–617], have very different chemical shifts in the two conformations, the most affected being Gln48(54) Hß2 (?d=1.53 ppm), which is placed directly above the Trp21(27) ring in the crystal structure of a3(VI). It is concluded that the origin of the multiple conformations of the central ß-sheet is a reorientation of the Trp21(27) ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant kM=1.01±0.05 s-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys14(20)-Cys38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3570–3582], is also likely to occur in a3(VI).

A 2D NMR experiment for assignment of backbone carbon resonances in small and medium-sized 15N-labelled proteins with 13C at natural abundance is presented. The experiment is a two-dimensional variant of the HNCO triple-resonance experiment and is demonstrated by application to a 6 kDa protein at relatively low concentration (2 mM) and temperature (30°C). The experiment is particularly suitable for assignment of carbonyl resonances.

A CO 2hydration activity for Mn(II) human carbonic anhydrase B (MnHCAB) of 7% of the activity of the native Zn 2+enzyme has been determined using a 13C magnetization—transfer NMR approach, that involves two complementary experiments. As this approach also allows a determination of the individual relaxation rates of the enzyme-bound CO 2and HCO −3, an evaluation could be made of the distances between these substrates and the paramagnetic Mn 2+in the active site. Thus HCO −3is found to bind directly to Mn 2+, whereas CO 2is attached relatively weakly to the enzyme without a direct bond to the metal ion.

A CO2hydration activity for Mn(II) human carbonic anhydrase B (MnHCAB) of 7% of the activity of the native Zn2+enzyme has been determined using a 13C magnetization—transfer NMR approach, that involves two complementary experiments. As this approach also allows a determination of the individual relaxation rates of the enzyme‐bound CO2and HCO−3, an evaluation could be made of the distances between these substrates and the paramagnetic Mn2+in the active site. Thus HCO−3is found to bind directly to Mn2+, whereas CO2is attached relatively weakly to the enzyme without a direct bond to the metal ion.

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhance ment-derived distance constraints, ten π torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 Å for the backbone and 1.92 Å for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well-defined, with r.m.s. deviations of 0.64 Å for the backbone and 1.51 Å for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in anintra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain α-helix. This interaction interferes with theinter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin – receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.

A new 2D NMR pulse sequence for E.COSY-type measurement of JHH coupling constants is introduced. It exploits a heteronuclear spin, e.g., 13C, for displacement in the ?1 frequency dimension via a large heteronuclear J coupling. The experiment is demonstrated by application to a heptapeptide at the natural abundance 13C level. It is suitable, for example, for measurement of 3JHH and 4JHH coupling constants in peptides and proteins.

Summary Linear prediction has been used to extrapolate the t1 domain of natural abundance1H-13C correlated two-dimensional (2D) FIDs of insulin. The FIDs were obtained by two different heteronuclear correlation experiments, one that utilizes heteronuclear multiple-quantum coherence during t1, and one that utilizes13C single-quantum coherence. It is shown that the enhancement of the resolution and sensitivity in the F1 dimension of the Fourier transform spectrum that results from the linear prediction extrapolation allows the t1 domain to be confined to a relatively short time period where the signal intensity is at maximum. In particular, it is found that the enhancement thus obtained is sufficiently good to allow an observation of the difference between the F1 line widths in the single-quantum and double-quantum coherence spectra.