Jürgen M. Schmidt

Simultaneous measurement of protein one-bond and two-bond nitrogen-carbon coupling constants using an internally referenced quantitative J-correlated [15N,1H]-TROSY-HNC experiment

A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone 1JNC′, 1JNCα, and 2JNCα coupling constants are obtained for the two [15N;13C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [2H;15N;13C]-labeled, large protein DFPase. A dependence of one-bond and two-bond JNCα values on protein backbone ψ torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated 15N-13C residual dipolar couplings.

Variation in protein C(alpha)-related one-bond J couplings.

Four types of polypeptide 1JCαX couplings are examined, involving the main‐chain carbon Cα and either of four possible substituents. A total 3105 values of 1JCαHα, 1JCαCβ, 1JCαC′, and 1JCαN′ were collected from six proteins, averaging 143.4 ± 3.3, 34.9 ± 2.5, 52.6 ± 0.9, and 10.7 ± 1.2 Hz, respectively. Analysis of variances (ANOVA) reveals a variety of factors impacting on 1J and ranks their relative statistical significance and importance to biomolecular NMR structure refinement. Accordingly, the spread in the 1J values is attributed, in equal proportions, to amino‐acid specific substituent patterns and to polypeptide‐chain geometry, specifically torsions ϕ, ψ, and χ1 circumjacent to Cα. The 1J coupling constants correlate with protein secondary structure. For α‐helical ϕ, ψ combinations, 1JCαHα is elevated by more than one standard deviation (147.8 Hz), while both 1JCαN′ and 1JCαCβ fall short of their grand means (9.5 and 33.7 Hz). Rare positive ϕ torsion angles in proteins exhibit concomitant small 1JCαHα and 1JCαN′ (138.4 and 9.6 Hz) and large 1JCαCβ (39.9 Hz) values. The 1JCαN′ coupling varies monotonously over the ϕ torsion range typical of β‐sheet secondary structure and is largest (13.3 Hz) for ϕ around − 160 . All four coupling types depend on ψ and thus help determine a torsion that is notoriously difficult to assess by traditional approaches using 3J. Influences on 1J stemming from protein secondary structure and other factors, such as amino‐acid composition, are largely independent. Copyright

Correlation of 2J couplings with protein secondary structure

Geminal two‐bond couplings (2J) in proteins were analyzed in terms of correlation with protein secondary structure. NMR coupling constants measured and evaluated for a total six proteins comprise 3999 values of 2JCαN′, 2JC′HN, 2JHNCα, 2JC′Cα, 2JHαC′, 2JHαCα, 2JCβC′, 2JN′Hα, 2JN′Cβ, and 2JN′C′, encompassing an aggregate 969 amino‐acid residues. A seamless chain of pattern comparisons across the spectrum datasets recorded allowed the absolute signs of all 2J coupling constants studied to be retrieved. Grouped by their mediating nucleus, C′, N′ or Cα, 2J couplings related to C′ and N′ depend significantly on ϕ,ψ torsion‐angle combinations. β turn types I, I′, II and II′, especially, can be distinguished on the basis of relative‐value patterns of 2JCαN′, 2JHNCα, 2JC′HN, and 2JHαC′. These coupling types also depend on planar or tetrahedral bond angles, whereas such dependences seem insignificant for other types. 2JHαCβ appears to depend on amino‐acid type only, showing negligible correlation with torsion‐angle geometry. Owing to its unusual properties, 2JCαN′ can be considered a “one‐bond” rather than two‐bond interaction, the allylic analog of 1JN′Cα, as it were. Of all protein J coupling types, 2JCαN′ exhibits the strongest dependence on molecular conformation, and among the 2J types, 2JHNCα comes second in terms of significance, yet was hitherto barely attended to in protein structure work. Proteins 2010.

One-bond and two-bond J couplings help annotate protein secondary-structure motifs: J-coupling indexing applied to human endoplasmic reticulum protein ERp18.

NMR coupling constants, both direct one‐bond (1J) and geminal two‐bond (2J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18 kDa protein comprise 1268 values of 1JCαHα, 1JCαCβ, 1JCαC′, 1JC′N′, 1JN′Cα, 1JN′HN, 2JCαN′, 2JHNCα, 2JC′HN, and 2JHαC′. Comparison with 1J and 2J data from reference proteins and pattern analysis on a per‐residue basis permitted main‐chain φ,ψ torsion‐angle combinations of many of the 149 amino‐acid residues in ERp18 to be narrowed to particular secondary‐structure motifs. J‐coupling indexing is here being developed on statistical criteria and used to devise a ternary grid for interpreting patterns of relative values of J. To account for the influence of the varying substituent pattern in different amino‐acid sidechains, a table of residue‐type specific threshold values was compiled for discriminating small, medium, and large categories of J. For the 15‐residue insertion that distinguishes the ERp18 fold from that of thioredoxin, the J‐coupling data hint at a succession of five isolated Type‐I β turns at progressively shorter sequence intervals, in agreement with the crystal structure. Proteins 2011.

Transforming between discrete and continuous angle distribution models: application to protein χ1 torsions

Two commonly employed angular-mobility models for describing amino-acid side-chain χ1 torsion conformation, the staggered-rotamer jump and the normal probability density, are discussed and performance differences in applications to scalar-coupling data interpretation highlighted. Both models differ in their distinct statistical concepts, representing discrete and continuous angle distributions, respectively. Circular statistics, introduced for describing torsion-angle distributions by using a universal circular order parameter central to all models, suggest another distribution of the continuous class, here referred to as the elliptic model. Characteristic of the elliptic model is that order parameter and circular variance form complementary moduli. Transformations between the parameter sets that describe the probability density functions underlying the different models are provided. Numerical aspects of parameter optimization are considered. The issues are typified by using a set of χ1 related 3J coupling constants available for FK506-binding protein. The discrete staggered-rotamer model is found generally to produce lower order parameters, implying elevated rotatory variability in the amino-acid side chains, whereas continuous models tend to give higher order parameters that suggest comparatively less variation in angle conformations. The differences perceived regarding angular mobility are attributed to conceptually different features inherent to the models.

Improved accuracy in measuring one-bond and two-bond 15N,13Cα coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

A bstractAn extension to HN(CO-α/β-N,Cα-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants 1JN′(i)Cα(i), 2JHN(i)Cα(i), 2JCα(i−1)N′(i), and 3JCα(i−1)HN(i) in 15N,13C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the 2JCαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both 2JCαN′ and 1JN′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.

Refinement of Protein Tertiary Structure by Using Spin-Spin Coupling Constants from Nuclear Magnetic Resonance Measurements

Modelling protein structure seems a challenging enterprise because the number of structure parameters required ordinarily exceeds the amount of independent data points available from experimental observations. Expressing the predominant conformation of a protein in terms of a geometry model, a polypeptide chain consisting of N atoms would command 3N – 6 Cartesian coordinates be fixed. Even for small proteins, this becomes a daunting number. Fortunately, so-called holonomic constraints limit the number of variables, leaving substantially fewer, truly relevant parameters for folding the polypeptide chain into its native tertiary structure. For example, adjusting bond lengths and the many angles between the covalent bonds connecting the atoms is of little concern and appropriate standard values can be inserted from tableworks (Pople & Gordon, 1967; Engh & Huber, 1991, 2006). Table 1 exemplifies for the 147-residue protein Desulfovibrio vulgaris flavodoxin how the number of truly independent internal rotational degrees of freedom amounts to less than one-tenth of the Cartesian coordinate set size...