The load-displacement behavior of 35 fresh adult cervical spine motion segments was measured in compression, shear, flexion, extension, lateral bending and axial torsion tests. Motion segments were tested both intact and with posterior elements removed. Applied forces ranged to 73.6 N in compression and to 39 N in shear, while applied moments ranged to 2.16 Nm. For each mode of loading, principal and coupled motions were measured and stiffnesses were calculated. The effect of disc degeneration on motion segment stiffnesses and the moments required for motion segment failure were also measured. In compression, the stiffnesses of the cervical motion segments were similar to those of thoracic and lumbar motion segments. In other modes of loading, cervical stiffnesses were considerably smaller than thoracic or lumbar stiffnesses. Removal of the posterior elements decreased cervical motion segment stiffnesses by as much as 50%. Degenerated cervical discs were less stiff in compression and stiffer in shear than less degenerated discs, but in bending or axial torsion, no statistically significant differences were evident. Bending moments causing failure were an order of magnitude lower than those for lumbar segments.
Can one protein sequence encode two structures? Oxidative folding of human insulin-like growth factor 1 (IGF-1), a globular protein of 70 residues, is shown to yield two products of similar thermodynamic stability. This observation is of particular interest in light of the recent demonstration that two of the three disulfide bonds in native IGF-1 rearrange in the presence of dithiothreitol [Hober, S., et al. (1992) Biochemistry 31, 1749-1756]. Kinetics of the IGF-1 folding pathway were monitored by high-performance liquid chromatography (rp-HPLC). Disulfide-pairing schemes of intermediates and products were established by peptide mapping. Two disulfide isomers were obtained as products: one with native insulin-like pairing [6-48; 18-61; 47-52] (designated native IGF-1; 60% yield) and the other with alternative pairing [6-47; 18-61; 48-52] (designated IGF-swap; 40% yield). The predominant early intermediate contains the single disulfide 18-61, which is shared in common by the two products. Relative yields of native IGF-1 and IGF-swap are independent of protein concentration under dilute conditions. In the absence of an added thiol reagent, each isomer is stable indefinitely at neutral pH; in the presence of an added thiol reagent, the two isomers interconvert with an Arrhenius activation barrier of 12 kcal/mol. Interconversion does not require complete reduction and yields the same ratio of products as initial folding, demonstrating thermodynamic control. Spectroscopic studies using circular dichroism (CD), infrared spectroscopy (FTIR), two-dimensional 1H-NMR (2D-NMR), and photochemical dynamic nuclear polarization (photo-CIDNP) suggest that IGF-1 and IGF-swap adopt similar secondary structures but distinct tertiary folds. Implications of these observations for understanding the topology of protein-folding pathways are discussed.
Preparations of the tetrodotoxin (TTX) and saxitoxin binding protein isolated from the electroplax of Electrophorus electricus are of high specific activity (greater than or equal to 2000 pmol of TTX binding sites/mg of protein) and appear to be homogeneous in that they contain only the large polypeptide previously identified to make up part of the voltage-sensitive sodium channel [Agnew, W. S., Moore, A. C., Levinson, S. R., & Raftery, M. S. (1980) Biochem. Biophys. Res. Commun. 92, 860-866]. This permits the inference that the TTX binding site, thought to be associated with the mouth of the ion channel, is located on this peptide. This peptide presumably corresponds to the large peptide, designated the alpha-peptide subunit, of the synaptosomal sodium channel [Hartshorne, R. P., & Catterall, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4620-4624]. No convincing evidence for lower molecular weight peptides has yet been found for the electroplax protein. A rapid and convenient method is described for preparation of milligram quantities of the pure, sodium dodecyl sulfate (NaDodSO4) denatured form of the peptide, and its amino acid and carbohydrate compositions are reported. The peptide behaved anomalously on NaDodSO4-polyacrylamide gels. It was demonstrated that the molecular weight cannot be accurately quantified by this method but that the true value likely exceeds the value of 260 000 reported previously. The denatured peptide displayed an electrophoretic microheterogeneity which may be ascribed to variations in bulky carbohydrate substituents and an extremely high free mobility which is inferred to result from binding of unusually large amounts of NaDodSO4.
Summary:To examine the loads imposed on the structures of the neck by the performance of physical tasks, a biomechanical model of the neck was constructed. The model incorporated 14 bilateral pairs of muscle equivalents crossing the C4 level. A double linear programming optimization scheme that minimized maximum muscle contraction intensity and then vertebral compression force while equilibrating external loads was used to calculate the muscle contraction forces required and the motion segment reactions produced by task performance. To test model validity, 14 healthy adult subjects performed a series of isometric tasks requiring use of their neck muscles. These tasks included exertions in attempted flexion, extension, and left and right lateral bending and twisting. Subjects exerted maximum and submaximurn voluntary efforts. During the performance, surface myoelectric activities were recorded at eight locations around the periphery of the neck at the C4 level. Calculated forces and measured myoelectric activities were then linearly correlated. Mean measured voluntary neck strengths in 10 male subjects were as large as 29.7 Nm. Four female subjects developed mean strengths that were approximately 60%-90% of those of the males. In both sexes, neck muscle strengths were approximately one order of magnitude lower than previously measured lumbar trunk strengths. Mean calculated neck muscle contraction forces ranged to 180 N. Mean calculated compression forces on the C4-5 motion segment ranged to 1164 N, lateral shear forces ranged to 125 N, and anteroposterior shear forces ranged to 135 N. Correlation coefficients between the calculated muscle forces and the measured myoelectric activities were as large as 0.85 in some muscles, but generally were smaller than this.
Insulin and insulin-related proteins contain three motif-specific disulfide bonds. Here we examine the role of these disulfide bonds in the folding and function of one family member, human insulin-like growth factor 1 (IGF-1). Analogues containing pariwise Cys-->Ser or Cys-->Ala substitutions were expressed in Escherichia coli, purified, and analyzed with respect to receptor-binding, solution structure, and thermodynamic stability. An analogue lacking all three disulfide bonds (designated des-Cys-IGF-1) is inactive and unfolded. Introduction of the [18-61] disulfide bond, previously shown to occur in an early intermediate in oxidative refolding [Miller, J. A., Owers-Narhi, L., Hua, Q. X., Rosenfeld, R., Arakawa, T., Rohde, M., Prestrelski, S., Lauren, S., S. Stoney, K. S., Tsai, L., & Weiss, M. A. (1993) Biochemistry (preceding paper in this issue)], results in a compact partially folded state with low but significant biological activity. Additional but incomplete structural organization and biological activity are observed following introduction of either the [6-48] or the [47-52] disulfide bonds. Native function, structure, and stability require the presence of all three disulfide bonds. These analogues provide genetic models of IGF-1 protein-folding intermediates. Their characterization suggests that bifurcation of the IGF-1 folding pathway reflects alternative late steps in the folding of a molten-globule intermediate.
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