Influence of the Zn(II) cofactor on the refolding of bovine carbonic anhydrase after denaturation with sodium dodecyl sulfate.

This paper uses capillary electrophoresis to follow a globular metalloprotein--bovine carbonic anhydrase II (BCA, EC 4.2.1.1)--on unfolding upon treatment with sodium dodecyl sulfate (SDS) and refolding upon removal of SDS, both in the presence and the absence of its Zn(II) cofactor. This research demonstrates that the Zn(II) cofactor is not required for refolding into a nativelike conformation, does not remain associated with the unfolded protein, and does not significantly change the rate of refolding. The presence of the Zn(II) cofactor, however, does increase the total amount of recovered protein by a factor of 2. Capillary electrophoresis could distinguish between native and denatured protein, based on the difference in electrophoretic mobility between the native protein and the aggregate of denatured protein and SDS. In addition, the active site was probed by observing binding of BCA to a charged arylsulfonamide using affinity capillary electrophoresis. These studies provide a foundation for future physical-organic studies using BCA as a model to examine interactions between proteins and SDS.

Proteomic analysis of hyperdynamic mouse hearts with enhanced sarcoplasmic reticulum calcium cycling.

Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.

Synthesis of monodisperse polymers from proteins.

Proteins are functional biopolymers; viewed as molecules, they are also monodisperse polyamides with chemically reactive side chains. This paper describes the use of proteins as starting materials for the synthesis of monodisperse polymers with nonbiological functionalities attached to the side chains. It demonstrates the complete derivatization of amine groups (lysine side chains and N-termini) on three different proteins by addition of activated carboxylate reagents in aqueous solutions containing sodium dedecyl sulfate (SDS), under denaturing conditions. Several different acylating reagents were used to generate derivatized proteins; the resulting compounds constitute a new class of monodisperse, semisynthetic polymers, having the potential for wide variation in the structure of the backbone and of the side chains. Modification of lysozyme on a gram scale demonstrated that the method can generate useful quantities of material.

Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis.

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.