Chemical shifts contain important site-specific information on the structure and dynamics of proteins. Deviations from statistical average values, known as random coil chemical shifts (RCCSs), are extensively used to infer these relationships. Unfortunately, the use of imprecise reference RCCSs leads to biased inference and obstructs the detection of subtle structural features. Here we present a new method, POTENCI, for the prediction of RCCSs that outperforms the currently most authoritative methods. POTENCI is parametrized using a large curated database of chemical shifts for protein segments with validated disorder; It takes pH and temperature explicitly into account, and includes sequence-dependent nearest and next-nearest neighbor corrections as well as second-order corrections. RCCS predictions with POTENCI show root-mean-square values that are lower by 25-78%, with the largest improvements observed for Hα andC'. It is demonstrated how POTENCI can be applied to analyze subtle deviations from RCCSs to detect small populations of residual structure in intrinsically disorder proteins that were not discernible before. POTENCI source code is available for download, or can be deployed from the URL http://www.protein-nmr.org .

Structural disorder is widespread in eukaryotic proteins and is vital for their function in diverse biological processes. It is therefore highly desirable to be able to predict the degree of order and disorder from amino acid sequence. It is, however, notoriously difficult to predict the degree of local flexibility within structured domains and the presence and nuances of localized rigidity within intrinsically disordered regions. To identify such instances, we used the CheZOD database, which encompasses accurate, balanced, and continuous-valued quantification of protein (dis)order at amino acid resolution based on NMR chemical shifts. To computationally forecast the spectrum of protein disorder in the most comprehensive manner possible, we constructed the sequence-based protein order/disorder predictor ODiNPred, trained on an expanded version of CheZOD. ODiNPred applies a deep neural network comprising 157 unique sequence features to 1325 protein sequences together with the experimental NMR chemical shift data. Cross-validation for 117 protein sequences shows that ODiNPred better predicts the continuous variation in order along the protein sequence, suggesting that contemporary predictors are limited by the quality of training data. The inclusion of evolutionary features reduces the performance gap between ODiNPred and its peers, but analysis shows that it retains greater accuracy for the more challenging prediction of intermediate disorder.

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Carbonyl13 C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13 C -13 C couplings on transverse relaxation measurements appears not to have been considered, and the potential to detect and quantify motions on the millisecond to microsecond time scale has not been fully explored. The present paper addresses these two issues. Simulations and experiments show that scalar couplings between adjacent backbone carbonyl carbon nuclei and between backbone and side-chain carbonyl/carboxyl carbon atoms in Asp and Asn residues interfere with the accurate determination of transverse relaxation rates by Carr-Purcell-Meiboom-Gill or on-resonance spin-lock measurements. The use of off-resonance radio-frequency fields avoids efficient cross-polarization, and offers a route towards accurate R 1r measurements. In addition, this approach yields dispersion in the transverse relaxation rate as a function of the effective field when conformational exchange is present. In the case of calcium-bound calbindin D 9k , 13 C off-resonance R 1r measurements yielded uniform values of R 2 along the polypeptide chain, indicating homogeneous chemical shift anisotropies and restricted dynamics on the picosecond to nanosecond time scale. Variation of R 2 as a function of the effective spin-lock field strength was not observed for any residue, indicating the absence of large-scale conformational changes of the protein backbone in the millisecond to microsecond time window. The absence of relaxation induced by internal motions on these wide-ranging time scales reinforces the view that calcium-loaded calbindin D 9k is extremely rigid. In contrast, for the C-terminal tryptic fragment of calmodulin containing the E140Q mutation we observed widespread exchange broadening. From the carbonyl transverse relaxation dispersion profile of Asp129 the exchange rate was determined to be 28 000 s −1 .

The g-gauche effect in 13 C NMR spectroscopy refers to the magnetic nonequivalence of the methyl carbons of the terminal isopropyl groups that are attached to branched alkanes; [1][2][3] this effect results in magnetic shielding of the carbon nucleus of the substituent in the gauche position by about 5 ppm relative to that of the same chemical group in the trans position. [4][5][6] Tonelli and colleagues demonstrated that the 13 C chemical shift of a carbon nucleus in a particular polymer stereoisomer is attributable solely to stereosequence-dependent differences in the probability that the given carbon atom is A C H T U N G T R E N N U N G involved in three-bond gauche interactions with other heavy atoms. Through this simple observation they were able to accurately and quantitatively predict the experimental 13 C NMR spectra of polypropylene and polypropylene model compounds with different stereoregularities. [4,5,7,8] We demonstrate here the use of the 13 C NMR g-gauche effect to establish leucine rotamer conformations in proteins, and provide a quantitative measure of their dynamics. This information is of value as a restraint on side-chain conformation in protein structure model building, [9,10] and is highly valuable for the interpretation of side chain methyl dynamics from 2 H or 13 C nuclear spin relaxation. [11][12][13][14][15][16] Although we focus below on methyl groups of leucine (Leu) residues, the g-gauche effect is a general determinant of chemical shifts of amino acid side-chain carbon nuclei, and expected to play an import role in their conformational analysis. [17][18][19] Leucine side chains can assume three stable staggered conformations of lowest potential energy as a function of the dihedral angle c 2 , referred to [20] as gauche(+) or p, gauche(À) or m, and trans or t (Figure 1). A carbon, rather than a proton, substituent in the gauche position leads to high internal energy, and results in the prevalence of conformations in which one atom is trans to the Ca atom, while the remaining atom is positioned gauche. Consequently, the two dominant c 2 rotamers are t and p. The energetics are mirrored by the c 2 side-chain distributions found in protein crystal structures. [20,21] In fact, at ambient temperature a small preference (2:1) of t over p is noticed in protein structures, as is observed for branched alkanes. [7] An analysis of the stereospecifically assigned methyl 13 C chemical shifts in the BioMagResBank (http://www.bmrb.wisc.edu/) is shown in Figure 2. The NMR data show that the preference of t over p observed in crystalline proteins is perfectly mirrored in solution.We have previously noted a strong correlation between the 3 J CC coupling and the methyl carbon chemical shift difference for leucine residues in two proteins; [22] this suggests that the wide distribution seen in Figure 2 could result from rotameric interconversion about the c 2 dihedral angle. A recent report by London and co-workers [19] supports this conclusion, and demonstrates that correlations between NMR side chain ...