Anion binding to a protein-protein complex lacks dependence on net charge

The binding of anions to proteins occurs in numerous physiological and metabolic processes. In an effort to understand the factors important in these interactions, we have studied the weak binding of phosphate and sulfate to a protein-protein complex using isothermal titration calorimetry. To our knowledge, this is the first system in which the thermodynamics of anion binding have been determined calorimetrically. By studying both phosphate and sulfate binding and using a range of pH values, the charge on the anion was varied from approximately -1 to -2. Surprisingly, no dependence of the binding energetics on the charge of the anion was observed. This result indicates that charge-charge interactions are not the dominant factor in binding and suggests the importance of hydrogen bonding in specifically recognizing and coordinating anions.     

Functional epitope on human neutrophil flavocytochrome b558

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.     

Evolutionary Landscapes for the Acquisition of New Ligand Recognition by RNA Aptamers

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro—unconstrained by confounding biological factors—is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences—defined as the sets functional tolerance for point mutations—and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.     

Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.     

Community outreach as an iterative dialogue among scientists and communities in the Texas gulf coast region

In response to significant environmental health challenges in Southeast Texas, a National Institute for Environmental Health Sciences Center at the University of Texas Medical Branch at Galveston was created to promote and conduct inter-disciplinary research in the areas of: (1) the molecular biology of DNA repair, replication and mutagenesis, (2) asthma pathogenesis in response to oxidative stress and viral exposures, and (3) environmental toxicant biotransformation. In addition, the NIEHS Center maintains close ties with neighboring communities through an active Community Outreach & Education Program (COEP) that develops and disseminates translational materials for use in environmental health awareness outreach, toxicology consultation, K-12 curriculum enrichment and in developing site-specific Community Partnership projects. The COEP core service divisions include: Environmental Arts & Sciences, Asthma Outreach & Education, Theater Outreach & Education, and Public Forum & Toxics Assistance. Public Forums focus on the use of Augusto Boal's Forum Theater dramaturgy to include the voices and local knowledge of communities within the process of Participatory Research. Forums create the preconditions for significant partnerships that link the hazardous risk perceptions and environmental health needs of communities with the expertise of NIEHS Center investigators and translational services provided through COEP outreach programs. The Forum process also creates leadership cores within environmentally challenged communities that facilitate the ongoing translational process and maintain the vital linkage between the health needs of communities and the analytic tools and the field and clinical technologies of the environmental sciences.     

Stabilization of proteins by ligand binding: application to drug screening and determination of unfolding energetics

The observed stability of a protein is altered when ligands bind, which results in a shift in the melting temperature (T(m)). Binding to the native state in the absence of binding to the denatured state will necessarily lead to an increase in the T(m), while binding to the unfolded state in the absence of native state binding will decrease the T(m) relative to that of the protein in the absence of ligand. These effects are required by the thermodynamics of reversible folding. However, the relationship between binding affinity and the magnitude of the observed temperature shift is not a simple correlation (i.e., a larger shift in T(m) does not necessarily mean tighter binding) and is complicated by interaction with the denatured state. Using exact simulations, the range of behavior for the dependence of the observed T(m) shift on the energetics of ligand binding is investigated here. Specifically, differential scanning calorimetry (DSC) curves are simulated for protein unfolding in the presence of ligands binding to both the native and denatured states. The results have implications for drug screening and the determination of heat capacity changes for protein unfolding.     

Frequency-dependent numerical dynamics in mosquitofish

Altering the genetic composition of a population can alter several aspects of its numerical dynamics. Whether natural populations routinely contain the genetic variation capable of affecting the stability of those dynamics is less clear. Here we report a study of experimental populations of mosquitofish (Gambusia holbrooki), designed to examine this issue. The experiment examined the numerical effects of varying the initial relative frequency of a rare male genotype. A higher relative frequency of the rare, melanic genotype produced higher mortality rates in melanic males, higher mortality rates in females, higher juvenile abundance, and fewer fluctuations in the numbers of females across time. This work demonstrates that a natural population can harbour genetic variants in a single gender that are capable of inducing qualitative differences in the numerical dynamics of the opposite gender, through the effects of negative frequency-dependent selection.     

Mutator dynamics in fluctuating environments

Populations with high mutation rates (mutator clones) are being found in increasing numbers of species, and a clear link is being established between the presence of mutator clones and drug resistance. Mutator clones exist despite the fact that in a constant environment most mutations are deleterious, with the spontaneous mutation rate generally held at a low value. This implies that mutator clones have an important role in the adaptation of organisms to changing environments. Our study examines how mutator dynamics vary according to the frequency of environmental fluctuations. Although recent studies have considered a single environmental switch, here we investigate mutator dynamics in a regularly varying environment, seeking to mimic conditions present, for example, under certain drug or pesticide regimes. Our model provides four significant new insights. First, the results demonstrate that mutators are most prevalent under intermediate rates of environmental change. When the environment oscillates more rapidly, mutators are unable to provide sufficient adaptability to keep pace with the frequent changes in selection pressure and, instead, a population of 'environmental generalists' dominates. Second, our findings reveal that mutator dynamics may be complex, exhibiting limit cycles and chaos. Third, we demonstrate that when each beneficial mutation provides a greater gain in fitness, mutators achieve higher densities in more rapidly fluctuating environments. Fourth, we find that mutators of intermediate strength reach higher densities than very weak or strong mutators.