Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.     

Effect of the (+)-CC-1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase.

(+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3-adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening [Lee, C.-S., Sun, D., Kizu, R., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 203-213]. In the studies described here, in which we have constructed site-directed DNA adducts on single-stranded DNA templates, we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead us to suspect that it is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes

The structures of NADP+ and magnesium isocitrate bound to the NADP(+)-dependent isocitrate dehydrogenase of Escherichia coli have been determined and refined at 2.5-A resolution. NADP+ is bound by the large domain of isocitrate dehydrogenase, a structure that has little similarity to the supersecondary structure of the nucleotide-binding domain of the lactate dehydrogenase-like family of nucleotide-binding proteins. The coenzyme-binding site confirms the fundamentally different evolution of the isocitrate dehydrogenase-like and the lactate dehydrogenase-like classes of nucleotide-binding proteins. In the magnesium-isocitrate complex, magnesium is coordinated to the alpha-carboxylate and alpha-hydroxyl oxygen of isocitrate in a manner suitable for stabilization of a negative charge on the hydroxyl oxygen during both the dehydrogenation and decarboxylation steps of the conversion of isocitrate to alpha-ketoglutarate. The metal ion is also coordinated by aspartate side chains 283' (of the second subunit of the dimer) and 307 and two water molecules in a roughly octahedral arrangement. On the basis of the geometry of the active site, the base functioning in the dehydrogenation step is most likely aspartate 283'. E. coli isocitrate dehydrogenase transfers a hydride stereospecifically to the A-side of NADP+, and models for a reactive ternary complex consistent with this stereospecificity are discussed.     

Population regulation in adult, but not juvenile, resident trout (Salmo trutta) in a Lake District stream

1. Unlike a neighbouring sea-trout population that showed strong density-dependent survival, a resident trout population ( Salmo trutta L.) showed simple proportionate survival in the early life-stages. However, this persistent population fluctuated within narrow limits. Mature adults, especially during spawning, were the only possible life-stage left in which regulation might occur. 2. An October census, just prior to spawning, was made at five sites (total area 300 m ² ) from 1965 to 1983. Gravel nests (redds) associated with females of known size were excavated outside these sites to obtain a power-function relationship between egg density per redd and female length (range 181–280 mm, n = 26). This relationship and the census data for females (range 186–284 mm) were used to estimate egg densities in each year-class. 3. The census data for the early life-stages (0+, 1+, 2+ trout) confirmed proportionate survival with no evidence for density-dependent regulation. In contrast, the number of spawning females produced in each year-class was strongly density dependent on the initial number of females that laid eggs at the start of the year-class. Similarly, total egg production in each year-class was density dependent on initial egg density. 4 Both relationships were well described by the Ricker and Beverton-Holt stock-recruitment models (P < 0.001) and the goodness-of-fit was similar for both models. This study is probably the first to provide clear evidence for fish population regulation in the adult, rather than the juvenile, stage.     

System controls on the aqueous distribution of mercury in the northern Florida Everglades

The forms and partitioning of aqueous mercury species in the canals and marshes of the Northern Florida Everglades exhibit strong spatial and temporal variability. In canals feeding Water Conservation Area (WCA) 2A, unfiltered total Hg (HgT_U) is less than 3 ng L^-1 and relatively constant. In contrast, methyl mercury (MeHg) exhibited a strong seasonal pattern, with highest levels entering WCA-2A marshes during July. Stagnation and reduced flows also lead to particle enrichment of MeHg. In the marshes of WCA-2A, 2B and 3A, HgT_U is usually <5 ng L^-1 with no consistent north–south patterns. However, for individual dates, aqueous unfiltered MeHg (MeHg_U) levels increase from north to south with generally lowest levels in the eutrophied regions of northern WCA-2A. A strong relationship between filtered Hg species and dissolved organic carbon (DOC), evident for rivers draining wetlands in Wisconsin, was not apparent in the Everglades, suggesting either differences in the binding sites of DOC between the two regions, or non-organic Hg complexation in the Everglades.     

Ca2+-dependent conformational changes in bovine GCAP-2.

GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent regulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fall in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP-2 is a small N-myristoylated protein containing four EF-hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-binding as measured by a Ca2+-dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP-2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N-terminus of Ca2+-bound GCAP-2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N-terminus of GCAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP-2 and recoverin, the structural consequences of Ca2+-binding for these two proteins are significantly dissimilar.     

Methionine-141 directly influences the binding of 4-methylpyrazole in human sigma sigma alcohol dehydrogenase

Pyrazole and its 4-alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human sigma sigma isoenzyme exhibits a 580-fold lower affinity for 4-methylpyrazole than does the human beta1beta1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4-methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human sigma sigma alcohol dehydrogenase (ADH) isoenzyme to 2.5 A resolution. Comparison of this structure to the human beta1beta1 isoenzyme structure indicated that the side-chain position for Met141 in sigma sigma ADH might interfere with 4-methylpyrazole binding. Mutation of Met141 in sigma sigma ADH to Leu (sigma141L) lowers the Ki for 4-methylpyrazole from 350 to 10 microM, while having a much smaller effect on the Ki for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate-binding pocket at a position close to the methyl group of 4-methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X-ray structure of the sigma141L mutant enzyme was determined to 2.4 A resolution. The three-dimensional structure of the mutant enzyme is identical to the wild-type enzyme, with the exception of the residue at position 141. Thus, the differences in 4-methylpyrazole binding between the mutant and wild-type sigma sigma ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class-specific differences in 4-methylpyrazole binding to the human ADH isoenzymes.     

Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.