Protein SEC

Peters, J.-M., Walsh, M. J., and Franke, W. W. An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec 18p and NSF. EMBOJ. 1990, 9, 1757-1767. 

The bridged complexes were prepared by (a) direct interaction of the two constituents or (b) one electron reduction of the fully oxidized bridged complex (it would be the Co "-Ru " complex in one example shown above ). The speed with which the reduction must be carried out depends on the subsequent electron transfer rate. Both chemical reductants or rapidly generated reducing radicals have been used. The latter approach (b) has been an effective one for investigating electron transfer within proteins (Sec. 5.9). A special approach (c) involves... 

Check the production level and compare the predicted molecular weights of each GST-fused protein sec Fig. 3b and Note 3). 

PPI source Folding protein (sec) Folding conditions Efficiency foldinjj peptide Ref."... 

Irreversible precipitation of proteins, called denaturation, is caused by heat, strong acids or bases, or various other agenis. Coagulation of egg white by heat, for example, is denaturation of the protein egg albumin. The extreme ease with which many proteins are denatured makes their study difficult. Denaturation causes a fundamental change in a protein, in particular destroying any physiological activity. (Denaturation appears to involve changes in the secondary structure of proteins, Sec. 36.16.)... 

Be able to compute the equilibrium state in biochemical reactions such as ligand binding and the denaturaiion (unfolding) of proteins (Secs. 15.4 and 15.5)... 

The protein to be translocated (called a pro-protein) is complexed in the cytoplasm with a chaperone (for example, the Sec B protein). The complex keeps the protein from folding prematurely, which would prevent it from passing through the secretory pore, which consists of two transmembrane proteins. Sec E and Sec Y. Sec A is an ATPase that helps drive the translocation. After the pro-protein is translocated, the leader peptide is cleaved by a membrane-bound protease and the protein can fold into its active three-dimensional form. 

Vibrational spectra of metalloporphyrins and related compounds have been studied extensively because of their biological importance as prosthetic groups of heme proteins (Sec. V-1 and V-2). Figure 111-13 shows the planar D4/, structure of a metalloporphyiin. 

The result of all these concurrent and often opposing effects is that an experimenter who wishes to use protein SEC data to support specific, quantitative conclusions concerning protein sizes and shapes is required to test multidimensional arrays of sets of conditions, rather than a one-dimensional array in which only the variable of specific interest is changed. 

The term preparative SEC encompasses all forms and scales of SEC depending on requirements for product. Preparative protein SEC has been categorized by the scale of the separation (70), which includes the following ... 

Table 2 Characteristics of Resins Currently Available for Preparative Protein SEC...

Although the phosphate buffer-NaCl system Is versatile and widely employed for nondenaturing protein SEC, the nonvolatile nature of the salts complicates subsequent concentration or buffer exchange of separated proteins. For these purposes we have employed a O.IM ammonium acetate buffer, pH 5.0, in place of the phosphate-NaCl buffer and observed equal resolution of the protein standards and virtually Identical values for individual proteins (Table 3). However, the ammonium acetate can be removed by lyophlllzatlon, thereby facilitating concentration of minute quantities of protein and/or... 

The volatile nature and ultraviolet transparency of the acetonitrlle-TFA eluant appear to make It an attractive candidate for protein SEC. However, we have found that a serious limitation of the system Is the low solubility of proteins of greater than about 15 kDa In 30-45% acetonitrile required for optimum resolution. This poor solubility results in severe protein aggregation problems, apparently limiting the usefulness of this system to peptides and very low molecular weight proteins. 

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