Penaeid and carid community changes in the St Lucia estuarine lake system,South Africa,under low water level,extended closed periods and marine reconnection conditions

Penaeid and carid communities were assessed in St Lucia over a wide range of saline conditions, water level conditions and mouth states, including a six-month marine connection and various connections to the Mfolozi Estuary. Samples were collected biannually in spring and autumn from November 2004 to May 2012 using seine nets. Seven penaeid and seven carid species were recorded, of which Palaemon pacificus, P. peringueyi, Penaeus indicus and Metapenaeus monoceros were the most abundant. The decline from 2004 to 2007 in both species recorded and densities of freshwater, estuarine and marine species during the initial closed period was linked to increased salinity, lack of recruitment and reduced estuarine surface area. Increased wave action and high seas following Cyclone Gamede opened the mouth in March 2007 after a 57-month closed period, resulting in recruitment of marine species, predominantly P. indicus, followed by a decline in densities after reclosure in August 2007. Increases in marine and freshwater species were evident after Mfolozi flood connections during 2008, 2009 and 2010. Highest densities were recorded after breaching in 2007 and after flood connections, highlighting the importance of a marine link to maintain the recruitment of penaeid species.     

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (Aβ). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (V_H) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic Aβ42 peptide segment (Aβ residues 17–42) in CDR3 on the solubility and conformational specificity of the corresponding V_H domains. We find that V_H expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, Aβ V_H domains with negatively charged CDR3 mutations show significant preference for recognizing Aβ fibrils relative to Aβ monomers, whereas the same V_H domains with other polar CDR3 mutations recognize both Aβ conformers. We observe similar behavior for a V_H domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8–37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.     

Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II

Crk-II is a signaling adaptor protein that is involved in many cellular processes including apoptosis, proliferation, and differentiation. It has a modular domain architecture consisting of an Src homology 2 domain (SH2) followed by two Src homology 3 (SH3) domains. The structures and ligand-binding properties of the SH2 and the middle SH3 domains are well-characterized. Several studies suggest that the C-terminal SH3 domain plays an important regulatory role in the protein; however, no structural information is available on this domain, and relatively little is known about its binding partners. In the current work, we have solved the solution NMR structure of the C-terminal SH3 domain. The domain adopts the standard SH3 fold comprising a five-stranded beta barrel. In agreement with alignment and modeling studies, the structure indicates that the canonical-binding surface of the SH3 domain is unusually polar and suggests that this domain may not bind typical PXXP ligands or that it may bind them with reduced affinity. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. These studies offer some insight into the likely structural and thermodynamic consequences of point mutations in the cSH3 domain that are known to deregulate Crk-II function. Our results set the stage for a better understanding the role of the cSH3 domain in the context of the full-length protein.     

The unfolded state of NTL9 is compact in the absence of denaturant

Interest in the unfolded state of proteins has grown with the realization that this state can have considerable structure in the absence of denaturants. Natively unfolded proteins, mutations that unfold proteins under native conditions, and changes in pH that induce unfolding are attractive models for the unfolded state in the absence of denaturant. The unfolded state of the N-terminal domain of ribosomal protein L9 (NTL9) was previously shown to contain significant non-native electrostatic interactions [Cho, J. H., Sato, S., and Raleigh, D. P. (2004) J. Mol. Biol. 338, 827-837]. NTL9 has a mixed alpha-beta structure and folds via a two-state mechanism. We have generated a model of the unfolded state of NTL9 in the absence of denaturant by substitution of an alanine for phenylalanine 5 located in the core of this protein. The CD spectrum of the variant, denoted as F5A, exhibits significantly less structure than the wild type; however, the mean residue ellipticity of F5A at 222 nm (-8200 deg cm(2) dmol(-)(1)) is considerably larger than expected for a fully unfolded protein, indicating that residual secondary structure is populated. F5A also has more residual structure than the urea-unfolded wild type. The stability of F5A is estimated to be at least 1 kcal/mol unfavorable, showing that the unfolded state is populated to 84% or more. NMR pulsed-field gradient measurements yield a hydrodynamic radius of 16.1 A for wild-type NTL9 and 20.8 A for the F5A variant in native buffer. The physiologically relevant unfolded state of wild-type NTL9 is likely to be even more compact than F5A since the mutation should reduce the level of hydrophobic clustering in the unfolded state in the absence of denaturant. The hydrodynamic radius of F5A increases to 25.9 A in 8 M urea, and a value of 23.5 A is obtained for the wild type under similar conditions. The results show that the unfolded state of F5A in the absence of denaturant is more compact and contains more structure than the urea-unfolded form.     

Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1

Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα_i1 and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα_i1 inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα_i-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα_i1. To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.     

Response of <Emphasis Type="Italic">Chrysaora quinquecirrha</Emphasis> medusae to low temperature

Because of their high abundance in Chesapeake Bay, Chrysaora quinquecirrha medusae may be an important reservoir of organic matter. The timing and location of the decomposition of biomass from medusae may have implications for carbon cycling in the bay. Our objective was to identify the cause of C. quinquecirrha medusa disappearance to better understand when and where decomposition occurs. A time series of visual surface counts and vertical net hauls in the Choptank River, a tributary of Chesapeake Bay, showed that as temperatures approached 15°C, C. quinquecirrha medusae disappeared from the surface, but persisted in net hauls until temperatures reached 10°C. In order to test whether medusae sink upon cooling, we exposed C. quinquecirrha medusae to low temperatures in large static tanks and measured their depth and pulsation rates twice daily for at least 6 days. This procedure was repeated three times through the 2008 jellyfish season. On average, individuals exposed to temperatures below 15°C were found deeper and pulsed slower than those in the warmer control tank. This suggests that low temperatures cause the medusae to sink before cooling to the limit of their physiological tolerance and may have implications for the deposition of organic matter associated with the seasonal disappearance of medusae from Chesapeake Bay.