Validity and reliability of a portable handheld dynamometer compared to a fixed isokinetic dynamometer to assess forearm torque strength.

To evaluate the effect of treatment on forearm rotation, torque muscle strength can be assessed using an isokinetic device (IKD) or a wrist dynamometer (WD). The aims of this study were 1) to determine concurrent validity and intra- and inter-rater reliability using the WD, and to examine correlations between WD and IKD in different positions; and 2) subsequently, to establish the intermethod reproducibility between WD as a handheld (HHD) or fixed device. We conducted a cross-sectional study in which torque strength was measured in healthy participants by two observers using an IKD and a WD. Study endpoints were concurrent validity (Pearson's r), intra- and inter-rater reliability, intermethod reproducibility (intraclass correlation coefficient: ICC) and measurement error (limits of agreement: LoA). Concurrent validity ranged, in the 2 studies assessing it, from r 0.37 to 0.52 for pronation and from r 0.50 to 0.82 for supination, with wide 95% confidence intervals. ICC for intra-rater reliability for pronation ranged from 0.85 to 0.91 and for supination from 0.91 to 0.95. ICC for inter-rater reliability for pronation ranged from 0.84 to 0.96 and for supination from 0.92 to 0.96. Despite the excellent intra- and inter-rater reliability and intermethod reproducibility for the WD-HHD and fixed WD, validity was low when compared to IKD and wide LoA indicated a high measurement error of approximately 20%. These results suggest that the WD cannot replace the IKD isometric mode for pronation and supination. LEVEL OF EVIDENCE: 2.

Potential bias in NMR relaxation data introduced by peak intensity analysis and curve fitting methods.

We present an evaluation of the accuracy and precision of relaxation rates calculated using a variety of methods, applied to data sets obtained for several very different protein systems. We show that common methods of data evaluation, such as the determination of peak heights and peak volumes, may be subject to bias, giving incorrect values for quantities such as R1 and R2. For example, one common method of peak-height determination, using a search routine to obtain the peak-height maximum in successive spectra, may be a source of significant systematic error in the relaxation rate. The alternative use of peak volumes or of a fixed coordinate position for the peak height in successive spectra gives more accurate results, particularly in cases where the signal/noise is low, but these methods have inherent problems of their own. For example, volumes are difficult to quantitate for overlapped peaks. We show that with any method of sampling the peak intensity, the choice of a 2- or 3-parameter equation to fit the exponential relaxation decay curves can dramatically affect both the accuracy and precision of the calculated relaxation rates. In general, a 2-parameter fit of relaxation decay curves is preferable. However, for very low intensity peaks a 3 parameter fit may be more appropriate.

Sequence-dependent correction of random coil NMR chemical shifts.

Random coil chemical shifts are commonly used to detect secondary structure elements in proteins in chemical shift index calculations. While this technique is very reliable for folded proteins, application to unfolded proteins reveals significant deviations from measured random coil shifts for certain nuclei. While some of these deviations can be ascribed to residual structure in the unfolded protein, others are clearly caused by local sequence effects. In particular, the amide nitrogen, amide proton, and carbonyl carbon chemical shifts are highly sensitive to the local amino acid sequence. We present a detailed, quantitative analysis of the effect of the 20 naturally occurring amino acids on the random coil shifts of (15)N(H), (1)H(N), and (13)CO resonances of neighboring residues, utilizing complete resonance assignments for a set of five-residue peptides Ac-G-G-X-G-G-NH(2). The work includes a validation of the concepts used to derive sequence-dependent correction factors for random coil chemical shifts, and a comprehensive tabulation of sequence-dependent correction factors that can be applied for amino acids up to two residues from a given position. This new set of correction factors will have important applications to folded proteins as well as to short, unstructured peptides and unfolded proteins.

Random coil chemical shifts in acidic 8 M urea: implementation of random coil shift data in NMRView.

Studies of proteins unfolded in acid or chemical denaturant can help in unraveling events during the earliest phases of protein folding. In order for meaningful comparisons to be made of residual structure in unfolded states, it is necessary to use random coil chemical shifts that are valid for the experimental system under study. We present a set of random coil chemical shifts obtained for model peptides under experimental conditions used in studies of denatured proteins. This new set, together with previously published data sets, has been incorporated into a software interface for NMRView, allowing selection of the random coil data set that fits the experimental conditions best.

Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.

The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system.

The region of the surface of the histidine-containing protein (HPr) which interacts with the A domain of the mannitol-specific Enzyme II (II(Amt1)) has been mapped by titrating the A-domain into a solution of 15N-labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty-five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of II(Amt1) while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of II(Amt1) resulted in chemical shift changes in a sub-set of the above residues; these were located more in the vicinity of the active site phospho-histidine. Phosphorylation of the HPr/II(Amt1) complex resulted in a HSQC spectrum which was indistinguishable from the P-HPr spectrum in the absence of II(Amt1) indicating that, as expected, the complex P-HPr/P-II(Amt1) does not exist even at the high concentrations necessary for NMR.

Conformation and mobility of the RNA-binding N-terminal part of the intact coat protein of cowpea chlorotic mottle virus. A two-dimensional proton nuclear magnetic resonance study.

Two-dimensional proton nuclear magnetic resonance (n.m.r.) experiments were performed on the coat protein of cowpea chlorotic mottle virus (molecular mass: 20.2 kDa) present as dimer (pH 7.5) or as capsid consisting of 180 protein monomers (pH 5.0). The spectra of both dimers and capsids showed resonances originating from the flexible N-terminal region of the protein. The complete resonance assignment of a synthetic pentacosapeptide representing this N terminus made it possible to interpret the spectra in detail. The capsid spectrum showed backbone amide proton resonances arising from the first eight residues having a flexible random coil conformation, and side-chain resonances arising from the first 25 N-terminal amino acids. The dimer spectrum showed also side-chain resonances of residues 26 to 33, which are flexible in the dimer but immobilized in the capsid. The n.m.r. experiments indicated that the conformation of the first 25 amino acids of the protein in dimers and capsids is comparable to the conformation of the synthetic peptide, which alternates among extended and helical conformations on the n.m.r. time-scale. It is suggested that the alpha-helical region, situated in the region between residues 10 and 20, binds to the RNA during assembly of the virus particle.