How uniform is the peptide plane geometry? A high-accuracy NMR study of dipolar Cα-C′/HN-N cross-correlated relaxation

Highly precise and accurate measurements of very small NMR cross-correlated relaxation rates, namely those between protein HiN-Ni and Ci−1α-Ci−1′ dipoles, are demonstrated with an error of 0.03s−1 for GB3. Because the projection angles between the two dipole vectors are very close to the magic angle the rates range only from −0.2 to +0.2s−1. Small changes of the average vector orientations have a dramatic impact on the relative values. The rates suggest deviation from idealized peptide plane geometry caused by twists around the C′-N bonds and/or pyramidalization of the nitrogen atoms. A clear alternating pattern along the sequence is observed in β strands 1, 3 and 4 of GB3, where the side chains of almost all residues with large positive rates are solvent exposed. In the α helix all rates are relatively large and positive. Some of the currently most accurate structures of GB3 determined by both high resolution X-ray crystallography and NMR are in satisfactory agreement with the experimental rates in the helix and β strand 3, but not in the loops and the two central strands of the sheet for which no alternating pattern is predicte

Mundart und Hochdeutsch im Vergleich

In der folgenden Darstellung geht es einerseits darum, an Beispielen aufzuzeigen, inwiefern die schweizerdeutschen Mundarten und die deutsche Standardsprache in Lautung, Formenbildung, Satzbau und Wortschatz auseinandergehen können, andererseits aber immer auch um das Aufweisen von Gemeinsamkeiten. Oft werden nämlich bestimmte Erscheinungen des dialektalen Sprachbaus vorschnell als Eigenarten der Mundart verstanden, obwohl dieselben Erscheinungen auch im gesprochenen Hochdeutschen anzutreffen sind. Somit liegen also häufig nicht Unterschiede zwischen Mundart und Standardsprache vor, sondern Unterschiede zwischen gesprochener Sprache und geschriebener Sprache. [vollständige Überarbeitung für eine zweite Auflage

TROSY experiment for refinement of backbone ψ and φ by simultaneous measurements of cross-correlated relaxation rates and 3,4 J HαHN coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential Hα-Cα/HN-N dipole/dipole and Hα-Cα/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the 3,4 J Hα HN coupling constants measured in an E.COSY manner. The conformational restraints for the ψ and φ angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [15N,13C]-DQ/ZQ-[15N,1H]-TROSY-E.COSY experiment to the 16kDa apo-form of the E.coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between ψ and φ angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformer

Side chain: backbone projections in aromatic and ASX residues from NMR cross-correlated relaxation

The measurements of cross-correlated relaxation rates between HN-N and Cβ-Cγ intraresidual and sequential dipolar interactions is demonstrated in ASN, ASP and aromatic residues. The experiment can be used for deuterated samples and no additional knowledge such as Karplus parametrizations is required for the analysis. The data constitutes a new type of information since no other method relates the Cβ-Cγ bond to HN-N. Using this method the dominant populations of rotamer states of χ1 can be readily cross checked provided that φ or ψ are known. In addition, dynamics on all timescales can be probed. As opposed to standard dynamics analysis of isolated bonds, the presented observables depend on relative dynamics with an interesting prospect to analyze correlated fluctuations of the two torsion angles φ or ψ with χ1. Experimental rates are compared to single conformer and ensemble representations of GB3 and ubiquitin. In particular, it is found that the recently published ubiquitin ensemble 2k39 improves the agreement obtained for 1UBQ. In general, however, input data restricting ASX and aromatic side chains in structure calculation is sparse highlighting the need for new NMR observable

Detection of C′,Cα correlations in proteins using a newtime- and sensitivity-optimal experiment

Sensitivity- and time-optimal experiment, called COCAINE (CO-CA In- and aNtiphase spectra with sensitivity Enhancement), is proposed to correlate chemical shifts of 13C′ and 13Cα spins in proteins. A comparison of the sensitivity and duration of the experiment with the corresponding theoretical unitary bounds shows that the COCAINE experiment achieves maximum possible transfer efficiency in the shortest possible time, and in this sense the sequence is optimal. Compared to the standard HSQC, the COCAINE experiment delivers a 2.7-fold gain in sensitivity. This newly proposed experiment can be used for assignment of backbone resonances in large deuterated proteins effectively bridging 13C′ and 13Cα resonances in adjacent amino acids. Due to the spin-state selection employed, the COCAINE experiment can also be used for efficient measurements of one-bond couplings (e.g. scalar and residual dipolar couplings) in any two-spin system (e.g. the N/H in the backbone of protein

Simultaneous 1H- or 2H-, 15N- and multiple-band-selective 13C-decoupling during acquisition in 13C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in 13C,13C-TOCSY spectra of uniformly deuterated and 13C, 15N-labeled protein and 13C,15N-labeled RNA samples is achieved by introduction of multiple-band-selective 13C-homodecoupling applied simultaneously with 1H- or 2H- and 15N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective 13C-decoupling in 2D-[13C,13C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear 13C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any 13C-detected experimen

Discrete Three-dimensional Representation of Macromolecular Motion from eNOE-based Ensemble Calculation

Three-dimensional structural data and description of dynamics are fundamental to infer and understand protein function. Structure determination by NMR follows well-established protocols while NMR relaxation phenomena provide insights into local molecular dynamics. However, methods to detect concerted motion were not pursued until very recently. Here, we present an ensemble-based structure determination protocol using ensemble-averaged distance restraints obtained from exact NOE (eNOE) rate constants. An application of our protocol to the model protein GB3 established an ensemble of structures that reveals correlated motion across the ?-sheet and concerted motion between the backbone and side chains localized in the core. Furthermore, the data repudiate concerted conformational exchange between the ?-sheet and the ?-helix

Stereospecific assignments in proteins using exact NOEs

Recently developed methods to measure distances in proteins with high accuracy by "exact” nuclear Overhauser effects (eNOEs) make it possible to determine stereospecific assignments, which are particularly important to fully exploit the accuracy of the eNOE distance measurements. Stereospecific assignments are determined by comparing the eNOE-derived distances to protein structure bundles calculated without stereospecific assignments, or an independently determined crystal structure. The absolute and relative CYANA target function difference upon swapping the stereospecific assignment of a diastereotopic group yields the respective stereospecific assignment. We applied the method to the eNOE data set that has recently been obtained for the third immunoglobulin-binding domain of protein G (GB3). The 884 eNOEs provide relevant data for 47 of the total of 75 diastereotopic groups. Stereospecific assignments could be established for 45 diastereotopic groups (96%) using the X-ray structure, or for 27 diastereotopic groups (57%) using structures calculated with the eNOE data set without stereospecific assignments, all of which are in agreement with those determined previously. The latter case is relevant for structure determinations based on eNOEs. The accuracy of the eNOE distance measurements is crucial for making stereospecific assignments because applying the same method to the traditional NOE data set for GB3 with imprecise upper distance bounds yields only 13 correct stereospecific assignments using the X-ray structure or 2 correct stereospecific assignments using NMR structures calculated without stereospecific assignment