Infrared spectra of the amino acid residues in H2O solution have been obtained in the 1800-1400-cm-1 region. It has been established that amino acid residues of arginine, asparagine, glutamine, aspartic and glutamic acids, lysine, tyrosine, histidine, and phenylalanine have intensive absorption in this spectral region. Infrared spectra for a set of model compounds have been measured. On the basis of these data, spectral parameters of amino acid residue absorption bands have been determined.

Denaturation of staphylococcal nuclease was studied in a temperature range from -7 to 70'C by scanning microcalorimetry and spectropolarimetry. It was found that the native protein is maximally stable at about 20'C and is denatured upon heating and cooling from this temperature. The heat and cold denaturation processes are approximated rather well by a two-state transition showing that the molecule is composed of a single cooperative system. The main difference between these two processes is in the sign of the enthalpy and entropy of denaturation: whereas the heat denaturation proceeds with increases in the enthalpy and entropy, the cold denaturation proceeds with decreases in both quantities. The inversion of the enthalpy sign occurs at about 15'C in an acetate buffer, but this temperature can be raised by addition of urea to the solvent.It is known that the denaturation of a protein is always accompanied by a significant increase in its enthalpy (for reviews, see refs. 1 and 2). The usually observed positive denaturational increment of heat capacity means that the enthalpy of protein denaturation is a temperature-dependent function. Thus, one can expect that the enthalpy of denaturation can, in principle, become zero and then even invert its sign at some low enough temperature, 7liV, changing from the factor stabilizing the native protein structure into a factor destabilizing this structure. Therefore, one can imagine that protein denaturation can occur not only upon heating but also upon cooling. In contrast with heat denaturation, which proceeds with heat absorption-i.e., an increase of enthalpy and entropy-cold denaturation should proceed with a release of heat-i.e., a decrease of enthalpy and entropy (3).The decrease of entropy upon the breakdown of the ordered native structure of protein molecules sounded like a paradox when it was first predicted more than 20 years ago by Brandts (4,5). As this prediction of cold denaturation was based on a long extrapolation of indirect data, there have been many attempts since then to confirm it by direct experiment. The first successful demonstration of cold denaturation was accomplished with myoglobin and apomyoglobin (3, 6).However, to show that cold denaturation is a general phenomenon specific for all globular proteins and not only for globins, it is highly desirable to demonstrate it on some other proteins. One of them was found to be lactate dehydrogenase on which cold denaturation was recently observed by Hatley and Franks (7). The other is staphylococcal nuclease (Nase), the denaturation of which is considered in this paper.Heat denaturation of Nase was studied calorimetrically by Calderon et al. (8) and it was shown that it is accompanied by a rather large increase of heat capacity, which indicated that cold denaturation of this protein might be observable under experimentally realizable conditions. MATERIALS AND METHODSThe Nase was prepared as described in detail by Calderon et al. (8) and was kindly supplied by John Gerlt (University of Maryland)...

Circular dichroic spectra of native, EDTA-treated and heat-denatured G-actin from chicken gizzard smooth muscle are virtually the same as those of rabbit skeletal muscle actin. The rates of changes produced by EDTA or heat in the secondary structure are, however, higher in the case of gizzard actin. Similar differences were found in the rates of inactivation as measured by loss of polymerizability during incubation with EDTA or Dowex 50. The results are explicable in terms of local differences in the conformation at specific site(s) important for maintaining the native state of actin monomer. Involvement of the ATP binding site was shown by measuring the equilibrium constant for the binding of ATP to the two actins. Difference in the conformation of some additional site(s) is indicated by a higher rate constant of inactivation of nucleotide-free actin observed for gizzard actin. No significant difference was found in the equilibrium constant for the binding of Ca2+ at the single high-affinity site in gizzard and skeletal muscle actin. Comparison of inactivation kinetics of actin from chicken gizzard, rabbit skeletal, bovine aorta, and bovine cardiac muscle suggests that the amino acid replacements Val-17 + Cys-17 and/ or Thr-89 + Ser-89 have a destabilizing effect on the native conformation of G-actin. The results indicate that deletion of the acidic residue at position 1 of the amino acid sequence has no effect on the conformation of the ATP binding site and the high-affinity site for divalent cation as well.It is well known that G-actin contains tightly bound cation and ATP in an amount of one mole of each per mole of the protein. If Mg2 + is not used for polymerization of actin during its purification, the bound cation in vitro is Ca2'. Both the bound Ca2+ and ATP are in an equilibrium with free Ca2+ and ATP in solution [I]. Although Ca2+ and ATP are probably bound at distinct sites in actin molecule [2, 31, their binding is interdependent: removal of either free Ca2+ or free ATP from the solution results in a release of both bound species [4 -111. This is followed by irreversible denaturation of actin accompanied by changes in its tertiary and secondary structure 112 -161. Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements have shown a decrease in the content of ordered structures upon denaturation by removal of the bound Ca" with EDTA or by heat treatment. Complete unfolding requires a treatment with urea or guanidinium hydrochloride [12, 151.We have recently observed [17] that G-actin from chicken gizzard smooth muscle undergoes a much faster spontaneous inactivation than rabbit skeletal muscle actin unless 0.1 -0.2 mM CaClz is present. In the present work the difference in apparent stability of these two actin isoforms has been studied in a more quantitative manner by comparing kinetics of their denaturation upon removal of the tightly bound Ca2+ by EDTA treatment and by measuring their heat-induced conformational transition. To determine which reaction(s) in the multi-step mechani...

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Evaluation of the secondary structure of caldesmon from c.d. spectra revealed that it contains 51% helix, 9% beta-strand and 40% of remainder structures. These values agree well with the predicted ones from amino acid sequence, assuming an extended chain structure for caldesmon. The estimates of the secondary-structure elements in C-terminal 34 kDa and 19 kDa fragments are: 11 and 12% helix, 22 and 20% beta-strand, 13 and 17% beta-turns and loops, and 54 and 50% of remainder structure respectively. The best fit of experimental data was obtained assuming the globular state of the fragments. On the basis of structural analysis and fragmentation by proteolytic and chemical cleavages the six-domain model of caldesmon is proposed.