Harald Schwalbe

The concept of a random coil: Residual structure in peptides and denatured proteins

Non-native states of proteins are of increasing interest because of their relevance to issues such as protein folding, translocation and stability. A framework for interpreting the wealth of experimental data for non-native states emerging from rapid advances in experimental techniques involves comparison with a random coll state, which possesses no structure except that inherent in the local interactions. We review here the concept of a random coil, from its global to its local properties. In particular, we focus on the description of a random coil in terms of statistical distributions in psi, phi space. We show that such a model, in combination with experimental data, provides insight into the structural properties of polypeptide chains and has significance for understanding protein folding and for molecular design.

A refined solution structure of hen lysozyme determined using residual dipolar coupling data.

A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H–15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low‐energy calculated structures has an average backbone RMSD of 0.50±0.13Å to the mean structure and of 1.49±0.10Å to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation‐dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior.

Chemical shifts in denatured proteins: Resonance assignments for denatured ubiquitin and comparisons with other denatured proteins

Chemical shift assignment is reported for the protein ubiquitin denatured in 8M urea at pHxa02. The variations in 15N chemical shifts of three different proteins (ubiquitin, disulfide reduced, carboxymethylated lysozyme, all-Ala-α-lactalbumin), all without disulfides and denatured in 8M urea at pHxa02 are compared to `random coil shifts of small model peptides (Braun etxa0al., 1994) and to the averaged native chemical shifts taken from the BMRB database. Both parameterizations show a remarkable agreement with the averaged measured 15N chemical shifts in the three denatured proteins. Detailed analysis of these experimental 15N chemical shifts provides an estimate of the influence of nearest neighbors and conformational preferences on the chemical shift and provides a direct means to identify non-random structural preferences in denatured proteins.

Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

19F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution 19F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution 19F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution mutants (Cys-140–Cys-316, Cys-65–Cys-316, and Cys-139–Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8–10 mg) in dodecylmaltoside and analyzed at 20°C by solution 19F NMR. Distinct chemical shifts were observed for all of the rhodopsin 19F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65–Cys-316 mutant suggests a close proximity between the two residues. When analyzed for 19F-19F NOEs, a moderate negative enhancement was observed for the Cys-65–Cys-316 pair and a strong negative enhancement was observed for the Cys-139–Cys-251 pair, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140–Cys-316 pair. These NOE effects demonstrate a solution 19F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.

Solution NMR spectroscopy of [α-15N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence

[α-15N]Lysine-labeled rhodopsin, prepared by expression of a synthetic gene in HEK293 cells, was investigated both by conventional and transverse relaxation optimized spectroscopy-type heteronuclear single quantum correlation spectroscopy. Whereas rhodopsin contains 11 lysines, 8 in cytoplasmic loops and 1 each in the C-terminal peptide sequence and the intradiscal and transmembrane domains, only a single sharp peak was observed in dodecyl maltoside micelles. This result did not change when dodecyl maltoside was replaced by octyl glucoside or octyl glucoside–phospholipid-mixed micelles. Additional signals of much lower and variable intensity appeared at temperatures above 20°C and under denaturing conditions. Application of the transverse relaxation optimized spectroscopy sequence resulted in sharpening of resonances but also losses of signal intensity. The single peak observed has been assigned to the C-terminal Lys-339 from the following lines of evidence. First, the signal is observed in HNCO spectra of rhodopsin, containing the labeled [13C]Ser-338/[15N]Lys-339 dipeptide. Second, addition of a monoclonal anti-rhodopsin antibody that binds to the C-terminal 8 aa of rhodopsin caused disappearance of the peak. Third, truncated rhodopsin lacking the C-terminal sequence Asp-330–Ala-348 showed no signal, whereas the enzymatically produced peptide fragment containing the above sequence showed the single peak. The results indicate motion in the backbone amide groups of rhodopsin at time scales depending on their location in the sequence. At the C terminus, conformational averaging occurs at the nanosecond time scale but varies from microsecond to millisecond in other parts of the primary sequence. The motions reflecting conformational exchange may be general for membrane proteins containing transmembrane helical bundles.

Determination of HN,H? and HN,C? coupling constants in 13C, 15N-labeled proteins

SummarySensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(HN,Hα) and 3J(HN,C′) coupling constants in uniformly 13C- and 15N-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the ϕ-angle for protein structure determination. The method is applied to uniformly 13C, 15N-labeled ribonuclease T1.

Millisecond Time Resolved Photo-CIDNP NMR Reveals a Non-Native Folding Intermediate on the Ion-Induced Refolding Pathway of Bovine α-Lactalbumin

Aspects of the structure of the intermediate populated after 200 ms in the Ca2+ -induced refolding of α-lactalbumin have been derived by time-resolved photo-CIDNP NMR methods. Refolding at constant denaturant concentration was initiated by laser-induced ion release from photolabile chelators. The NMR data demonstrated that part of the polypeptide chain in the β-domain of α-lactalbumin samples adopt non-native conformations while a hydrophobic core of the α-domain is already formed.

On the Solution Structure of PHB: Preparation and NMR Analysis of Isotopically Labeled Oligo[(R)-3-hydroxybutanoic Acids] (OHBs)

While the chain conformation of poly- and oligo[(R)-3-hydroxybutanoate] (PHB, OHB) is known to be 21- and 31-helical in stretched fibers and in the crystalline state, respectively (Fig.u20052), the structure in solution is unknown. To be able to determine the NMR-solution structure, specifically labeled linear oligomers have been prepared: a 16-mer consisting of alternating pairs of fully 13C-labeled and non-labeled residues (1) and a 20-mer containing an O-13CH(13CH2D)-13CHDSi-13CO residue in position 9 (from the O-terminus) and a fully 13C-labeled residue in position 12 (2), both with (t-Bu)Ph2Si protection at the O- and Bn protection at the C-terminus. The labeled (R)-3-hydroxybutanoic acid building blocks were prepared by Noyori hydrogenation of the ethyl ester of fully 13C-labeled acetoacetic acid, and the D-atoms were incorporated by D2/Pd-C reduction of a previously reported dibromo-1,3-dioxinone 8 (Schemeu20051). The oligomers were obtained by a series of fragment couplings (Schemesu20052 and 3). 600-MHz NMR COSY, HSQC, ROESY, and cross-correlated relaxation measurements (Figs.u20054u2009–u20096, 9, and 12, and Tablesu20051u2009–u20093) at different temperatures and interpretations thereof led to assignments of all resonances, including those from the diastereotopic C(2)H2 protons, and to determination of the conformationally averaged dihedral angles ϕ2 and ϕ3 (Figs.u20052, 7, and 8) in the chain of the oligoester. The conclusions are: all but five or six terminal residues adopt the same conformation; the 21 helix is not the predominant secondary structure; the structure of the HB chain is averaged, even at –30°. Our investigation confirms the high flexibility of the polyester chain, a property that has been deduced previously from biological studies of PHB in membranes, in ion channels, and as appendage of proteins.

Determination of sugar conformation in large RNA oligonucleotides from analysis of dipole–dipole cross correlated relaxation by solution NMR spectroscopy

A new experiment, the forward directed quantitative Γ-HCCH-TOCSY for the measurement of the conformation of the five-membered ribosyl unit in RNA oligonucleotides, is presented. The experiment relies on quantification of cross peak intensities caused by evolution of CH,CH-dipole–dipole cross correlated relaxation in non-evolution periods and the resolution enhancement obtainable in forward directed HCC-TOCSY transfer. Cross correlated relaxation rates are interpreted to reveal the sugar conformation of 22 out of 25 nucleotides in an isotopically labelled 25-mer RNA. The results obtained with this new method are in agreement with the conformational analysis derived from 3J(H,H) coupling constants.

Comparison of the NMR spectroscopy solution structures of pyranosyl-RNA and its nucleo-δ-peptide analogue

The design of polymers that could mimic biomolecules in their ability to form assemblies similar to ribo‐ and deoxyribonucleic acids has become an attractive field of chemical research, and NMR spectroscopy has played a vital role in the determination of the three‐dimensional structure of these newly designed nonnatural polymers. The structure of a self‐complementary octamer duplex of pyranosyl‐RNA (pRNA) has been determined by using NMR spectroscopy experimental data and an Xplor structure calculation protocol. The structure has been compared with the structure of a duplex formed by a designed nucleo‐δ‐peptide analogue of pRNA. The two duplexes assume one predominant conformation and show a high structural similarity. The conformation type of both structures agrees with those predicted based on qualitative conformational analysis and both structures show a good convergence toward the average torsion angles derived by NMR spectroscopy.