Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li 1 , Na 1 , K 1 , Rb 1 , Cs 1 , GdnH 1 , and Ca 21 ), identity. The Q* decreased in the order, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO 2À 4 anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.

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A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.

The fang-like jaws of the marine polychaete Nereis Virens possess remarkable mechanical properties considering their high protein content and lack of mineralization. Hardness and stiffness properties in the jaw tip are comparable to human dentin and are achieved by extensive coordination of Zn 2+ by a histidine-rich protein framework. In the present study, the predominant protein in the jaw tip, NVjp-1, was purified and characterized by partial peptide mapping and molecular cloning of a partial cDNA from a jaw pulp library. The deduced amino acid sequence revealed an ∼38 kDa histidine-rich protein rich in glycine and histidine (∼36 and 27%, respectively) with no well-defined repetitive motifs. The effects of pH and metal treatment on aggregation, secondary structure, and hydrodynamic properties of recombinant Nvjp-1 are described. Notably, Zn treatment induced the formation of amyloid-like fibers.

Spiders have captured the interest of scientists for many years because spider silks are among the toughest materials, having properties that surpass some man-made synthetic materials. Spinning recombinant silk to duplicate those properties has proved to be extremely difficult. This is the first known report of spinning recombinant silk fibers in an aqueous environment. The method seeks to keep the protein soluble throughout the process, not unlike the way the spider stores and spins silk. Recombinant silk proteins were produced by bacterial fermentation in which the cell pellets were lyophilized and lysed with organic acid. Silk protein was purified from the lysate by chromatography and processed in dilute denaturing buffer into a fiber spinning solution. Circular dichroism measurements of the silk solutions revealed an increase in β-sheet content as a function of time. Time-dependent self-association of silk protein was monitored in solution by dynamic light scattering. Furthermore, the observed increase in β-sheet content and self-association appear to be required for fiber formation. Recombinant silk fibers were 10−60 μm in diameter, water insoluble, and birefringent, indicating molecular orientation within the fiber.

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The fast, efficient, and accurate release of proteins from cells and tissues is a critically important initial step in most analytical processes and is essential to reliable proteomic analyses. Two-dimensional gel electrophoresis (2DGE) 1 can be an accurate representation of a proteome only if the entire protein constituency of cells is recovered during the sample preparation process. Pressure cycling technology (PCT) uses alternating cycles of high and low hydrostatic pressure to effectively induce the lysis of cells and tissues in preparation for 2DGE and other analytical or preparative methods. Rapid cycling between high and low pressure is more disruptive than high pressure alone, as evidenced by the increased protein yields from Saccharomyces cerevisae correlating to the number of pressure cycles rather than the total elapsed time at high pressure [1]. Similarly, Herrero and coworkers [2] reported a 20% increase in phycobiliproteins yielded from Spirulina platensis when multiple iterations of a pressurized liquid extraction method were performed. Previously, Geiser and coworkers [3] reported the release of 37% more protein from the nematode Caenorhabditis elegans by PCT than by sonication. From gram-negative bacteria, PCT reportedly yielded 14.2% more protein from Escherichia coli than did bead beating [4,5] and yielded 17.1% more protein from Rhodopseudomonas palustris than did enzymatic lysis with lysozyme [6]. For mammalian tissues, PCT also isolated more protein from liver, including several unique proteins that were not isolated by conventional homogenization techniques [7]. From adipose tissue, PCT extracted more protein than did pulverization under liquid nitrogen and detergent extraction of the triturate. The Barocycler NEP-3229 instrument, disposable polypropylene PULSE Tubes FT-500, and ProteoSOLVE IEF Reagent were obtained from Pressure BioSciences (West Bridgewater, MA, USA). Linear immobilized pH gradients (IPGs, pH 3-10) were obtained from Proteome Systems (Woburn, MA, USA). Nonlinear IPGs and PDQuest version 7.1 image analysis software were obtained from Bio-Rad (Hercules, CA, USA). Ultrafree-CL