Evaluation of Backbone Proton Positions and Dynamics in a Small Protein by Liquid Crystal NMR Spectroscopy

Abstract

NMR measurements of a large set of protein backbone one-bond dipolar couplings have been carried out to refine the structure of the third IgG-binding domain of Protein G (GB3), previously solved by X-ray crystallography at a resolution of 1.1 A. Besides the commonly used bicelle, poly(ethylene glycol), and filamentous phage liquid crystalline media, dipolar couplings were also measured when the protein was aligned inside either positively or negatively charged stretched acrylamide gels. Refinement of the GB3 crystal structure against the (13)C(alpha)-(13)C' and (13)C'-(15)N dipolar couplings improves the agreement between experimental and predicted (15)N-(1)H(N) as well as (13)C(alpha)-(1)H(alpha) dipolar couplings. Evaluation of the peptide bond N-H orientations shows a weak anticorrelation between the deviation of the peptide bond torsion angle omega from 180 degrees and the angle between the N-H vector and the C'-N-C(alpha) plane. The slope of this correlation is -1, indicating that, on average, pyramidalization of the peptide N contributes to small deviations from peptide bond planarity (<omega> = 179.3 +/- 3.1 degrees ) to the same degree as true twisting around the C'-N bond. Although hydrogens are commonly built onto crystal structures assuming the N-H vector orientation falls on the line bisecting the C'-N-C(alpha) angle, a better approximation adjusts the C(alpha)-C'-N-H torsion angle to -2 degrees. The (15)N-(1)H(N) dipolar data do not contradict the commonly accepted motional model where angular fluctuations of the N-H bond orthogonal to the peptide plane are larger than in-plane motions, but the amplitude of angular fluctuations orthogonal the C(alpha)(i-1)-N(i)-C(alpha)(i) plane exceeds that of in-plane motions by at most 10-15 degrees. Dipolar coupling analysis indicates that for most of the GB3 backbone, the amide order parameters, S, are highly homogeneous and vary by less than +/-7%. Evaluation of the H(alpha) proton positions indicates that the average C(alpha)-H(alpha) vector orientation deviates by less than 1 degrees from the direction that makes ideal tetrahedral angles with the C(alpha)-C(beta) and C(alpha)-N vectors.

Correlated motions are a fundamental property of β-sheets

“…1b, we provide a summary of the correlation analysis, where the crankshaft motion, a rotation of the peptide plane about the C a i À 1 -C a i axis that leads to the anti-correlation of c i À 1 and f i often observed in MD simulations, is shown in italics 24 . The crankshaft motion is equivalent to that put forward in the onedimensional Gaussian axial fluctuation model (1D-GAF) of the protein backbone and to the g motion of the related 3D-GAF motional model, the amplitudes of which have been extensively studied by NMR spectroscopy 20,21,[25][26][27] . The anti-correlation is due to the rigidity of the peptide plane, which couples the motion Figure 1 | b-sheet ensemble correlations observed within and between the strands.…”

Protein dynamics and function from solution state NMR spectroscopy

“…a multitude of different structural states are sampled in absence of target proteins and the presence of a specific target can select a specific ubiquitin structure. Finally, it is important to note that while this section focused on EROS, several other approaches for interpreting dynamics on the basis of RDCs exist (Bouvignies et al 2006(Bouvignies et al , 2007Tolman, 2002;Tolman et al 2001;Ulmer et al 2003).…”

ProCS15: A DFT-based chemical shift predictor for backbone and C β atoms in proteins

“…The structures are geometry optimized using PM6-D3H+ (Kromann et al, 2014) using the PCM solvation model (Tomasi et al, 2005;Steinmann et al, 2013) and the CHARMM22/CMAP force field (Mackerell, 2004) using the GB/SA solvation model (Qiu et al, 1997) with the 1UBQ (Vijay- Kumar et al, 1987) and 2OED (Ulmer et al, 2003) structures as starting points. The PM6-D3H+ optimizations are done using the GAMESS program (Schmidt et al, 1993) with a convergence criterion of 5 × 10 −4 atomic units, while the CHARMM22/CMAP optimizations are done using TINKER (Ponder and Richards, 1987) with the default convergence criterion of 0.01 kcal/mole/Å.…”