The conformational changes of ribonuclease A due to thermal and guanidine hydrochloride denaturation were monitored by means of precise density and sound velocity measurements. It was found that the apparent molar volume decreased but the adiabatic compressibility increased on thermal denaturation under acidic conditions (pHs 1.60, 1.90, and 2.08). On the other hand, guanidine hydrochloride denaturation (pH 2.00) brought about large decreases in the compressibility and apparent molar volume. These results indicate that the conformation of the denatured protein is greatly different between the two types of denaturation: the thermally denatured state corresponds to the structure with enhanced thermal fluctuation having a residual secondary structure and a high local concentration of nonpolar groups exposed, but the guanidine hydrochloride denaturation leads to exposure of a large amount of amino acid residues, resulting in an increase in hydration and a decrease in the internal cavity. The compressibility changes due to both types of denaturation were not correlated to a loss of the secondary structure, as judged by means of circular dichroism. These findings suggest that the compactness and thermal fluctuation of the protein cannot be described by a two-state denaturation model and that there are some molten-globule-like intermediates in the denaturation processes.

SUMMARY1. A technique has been developed with which the stiffness changes in frog skeletal muscle can be continuously recorded by measuring the propagation velocity of ultrasonic waves (3-7 MHz) with negligibly small perturbations to the contractile system.2. The resting muscle stiffness was 2 256+00002 x 109 N/m2 (S.D.) at 1-2°C (n = 10) and 2-480+0-007 x 109 N/M2 at 19-20°C (n = 12) in the longitudinal direction, and 2-223 + 04008 x 109 N/m2 at 1-2°C (n = 8) and 2 437 + 0 007 x 109 N/M2 at 19-20°C (n = 9) in the transverse direction.3. The resting muscle stiffness measured with ultrasonic waves was virtually insensitive to the resting force development, i.e. the extension of the parallel elastic component.4. The longitudinal muscle stiffness increased during isometric contraction at a rate faster than the force development. The amount of increase of the longitudinal stiffness in an isometric tetanus at 2 2 ,um sarcomere length was 24 ±+01 x 107 N/M2 at 1-2°C (n = 10) and 6-5 + 1-3 x 107 N/m2 at 19-20°C (n = 12).5. On the other hand, the transverse muscle stiffness decreased during isometric contraction at a rate faster than the force development. The amount of decrease of the transverse stiffness in an isometric tetanus at 22 jtm sarcomere length was 5-6+01 x 107 N/M2 at 1-2°C (n = 8) and 64+0-3 x 107 N/M2 at 19-20°C (n = 9).6. The amount of both the longitudinal and the transverse stiffness changes during an isometric tetanus decreased linearly with increasing sarcomere length, indicating that the stiffness changes during contraction reflect the formation of cross-links between the myofilaments.7. Both the longitudinal and the transverse stiffness increased when resting muscle was put into rigor state. The rigor muscle stiffness was insensitive to small stretches, i.e. the strain of the rigor cross-links.8. These results are discussed in connection with the behaviour of cross-bridges during isometric contraction and in rigor.

The adiabatic compressibility (βs) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT). Interestingly, the βs values of both DHFR and AspAT were influenced markedly by the mutations at glycine‐121 and valine‐39, respectively, in which the magnitude of the change was proportional to the enzyme activity. This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function. This is the first report on the compressibility of mutant proteins.

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