Calsequestrin, the major Ca2+ storage protein of muscle, coordinately binds and releases 40-50 Ca2+ ions per molecule for each contraction-relaxation cycle by an uncertain mechanism. We have determined the structure of rabbit skeletal muscle calsequestrin. Three very negative thioredoxin-like domains surround a hydrophilic center. Each monomer makes two extensive dimerization contacts, both of which involve the approach of many negative groups. This structure suggests a mechanism by which calsequestrin may achieve high capacity Ca2+ binding. The suggested mechanism involves Ca2+-induced collapse of the three domains and polymerization of calsequestrin monomers arising from three factors: N-terminal arm exchange, helix-helix contacts and Ca2+ cross bridges. This proposed structure-based mechanism accounts for the observed coupling of high capacity Ca2+ binding with protein precipitation.

A Fourier transform infrared spectroscopic method has been developed to analyze protein secondary structure by employing the amide III spectral region (1350–1200 cm−1)· Benefits of using the amide III region have been shown to be substantial. The interference from the water vibration (∼1640 cm−1) in the amide I region can be avoided when one is using the amide III band; furthermore, the amide III region also presents a more characterized spectral feature which provides easily resolved and better defined bands for quantitative analysis. Estimates of secondary structure are accomplished with the use of Fourier self-deconvolution, second derivatization, and curve-fitting on original protein spectra. The secondary structure frequency windows (α-helix, 1328–1289 cm−1; unordered, 1288–1256 cm−1; and β-sheets, 1255–1224 cm−1) have been obtained, and estimates of secondary structural contents are consistent with X-ray crystallography data for model proteins and parallel results obtained with the use of the amide I region. We have further applied the analysis to the structural change of calsequestrin upon Ca2+ binding. Treatment of calsequestrin with 1 mM Ca2+ results in the formation of crystalline aggregates accompanied by a 10% increase in α-helical structure, which is consistent with previous results obtained by Raman spectroscopy. Thus the amide III region of protein IR spectra appears to be a valuable tool in estimating individual protein secondary structural contents.

We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureusmastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureuscells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalizationS. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes.