Corey J. Wilson

The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding

Abstract.In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli [1]. This proposal was followed by an explication of allosteric behavior by Monod and colleagues [2]. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

Role of cofactors in metalloprotein folding

Metals are commonly found as natural constituents of proteins. Since many such metals can interact specifically with their corresponding unfolded proteins in vitro , cofactor-binding prior to polypeptide folding may be a biological path to active metalloproteins. By interacting with the unfolded polypeptide, the metal may create local structure that initiates and directs the polypeptide-folding process. Here, we review recent literature that addresses the involvement of metals in protein-folding reactions in vitro . To date, the best characterized systems are simple one such as blue-copper proteins, heme-binding proteins, iron-sulfur-cluster proteins and synthetic metallopeptides. Taken together, the available data demonstrates that metals can play diverse roles: it is clear that many cofactors bind before polypeptide folding and influence the reaction; yet, some do not bind until a well-structured active site is formed. The significance of characterizing the effects of metals on protein conformational changes is underscored by the many human diseases that are directly linked to anomalous protein-metal interactions.

Adsorption of multimeric T cell antigens on carbon nanotubes: effect on protein structure and antigen-specific T cell stimulation.

Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin.

Experimental Evolution of Adenylate Kinase Reveals Contrasting Strategies toward Protein Thermostability

Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK(BSUB) Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK(BSUB) G213E decreases protein stability by >25 degrees C, the same mutation in the background of AK(BSUB) Q199R increases stability by 3.4 degrees C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK(BSUB) double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.

Understanding Thermal Adaptation of Enzymes through the Multistate Rational Design and Stability Prediction of 100 Adenylate Kinases

Careful balance between structural stability and flexibility is a hallmark of enzymatic function, and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Herein, we describe the rational design of 100 temperature-adapted adenylate kinase enzymes using a multistate design strategy that incorporates the impact of conformational changes to backbone structure and stability, in addition to experimental analysis of thermostability and function. Comparison of the experimental temperature of maximum activity to the melting temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data were used to produce accurate predictions of thermostability, providing the requisite complement for de novo temperature-adapted enzyme design. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of the thermodynamic and structural limits of function.

Effect of Chemical Oxidation on the Sorption Tendency of Dissolved Organic Matter to a Model Hydrophobic Surface

The application of chemical oxidants may alter the sorption properties of dissolved organic matter (DOM), such as humic and fulvic acids, proteins, polysaccharides, and lipids, affecting their fate in water treatment processes, including attachment to other organic components, activated carbon, and membranes (e.g., organic fouling). Similar reactions with chlorine (HOCl) and bromine (HOBr) produced at inflammatory sites in vivo affect the fate of biomolecules (e.g., protein aggregation). In this study, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to evaluate changes in the noncovalent interactions of proteins, polysaccharides, fatty acids, and humic and fulvic acids with a model hydrophobic surface as a function of increasing doses of HOCl, HOBr, and ozone (O3). All three oxidants enhanced the sorption tendency of proteins to the hydrophobic surface at low doses but reduced their sorption tendency at high doses. All three oxidants reduced the sorption tendency of polysaccharides and fatty acids to the hydrophobic surface. HOCl and HOBr increased the sorption tendency of humic and fulvic acids to the hydrophobic surface with maxima at moderate doses, while O3 decreased their sorption tendency. The behavior observed with two water samples was similar to that observed with humic and fulvic acids, pointing to the importance of these constituents. For chlorination, the highest sorption tendency to the hydrophobic surface was observed within the range of doses typically applied during water treatment. These results suggest that ozone pretreatment would minimize membrane fouling by DOM, while chlorine pretreatment would promote DOM removal by activated carbon.

Role of lysine during protein modification by HOCl and HOBr: halogen-transfer agent or sacrificial antioxidant?

Although protein degradation by neutrophil-derived hypochlorous acid (HOCl) and eosinophil-derived hypobromous acid (HOBr) can contribute to the inactivation of pathogens, collateral damage to host proteins can also occur and has been associated with inflammatory diseases ranging from arthritis to atherosclerosis. Though previous research suggested halotyrosines as biomarkers of protein damage and lysine as a mediator of the transfer of a halogen to tyrosine, these reactions within whole proteins are poorly understood. Herein, reactions of HOCl and HOBr with three well-characterized proteins [adenylate kinase (ADK), ribose binding protein, and bovine serum albumin] were characterized. Three assessments of oxidative modifications were evaluated for each of the proteins: (1) covalent modification of electron-rich amino acids (assessed via liquid chromatography and tandem mass spectrometry), (2) attenuation of secondary structure (via circular dichroism), and (3) fragmentation of protein backbones (via sodium dodecyl sulfate-polyacrylamide gel electrophoresis). In addition to forming halotyrosines, HOCl and HOBr converted lysine into lysine nitrile (2-amino-5-cyanopentanoic acid), a relatively stable and largely overlooked product, in yields of up to 80%. At uniform oxidant levels, fragmentation and loss of secondary structure correlated with protein size. To further examine the role of lysine, a lysine-free ADK variant was rationally designed. The absence of lysine increased yields of chlorinated tyrosines and decreased yields of brominated tyrosines following treatments with HOCl and HOBr, respectively, without influencing the susceptibility of ADK to HOX-mediated losses of secondary structure. These findings suggest that lysine serves predominantly as a sacrificial antioxidant (via formation of lysine nitrile) toward HOCl and as a halogen-transfer mediator [via reactions involving ε-N-(di)haloamines] with HOBr.

Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin

Understanding how the folding of proteins establishes their functional characteristics at the molecular level challenges both theorists and experimentalists. The simplest test beds for confronting this issue are provided by electron transfer proteins. The environment provided by the folded protein to the cofactor tunes the metals electron transport capabilities as envisioned in the entatic hypothesis. To see how the entatic state is achieved one must study how the folding landscape affects and in turn is affected by the metal. Here, we develop a coarse-grained functional to explicitly model how the coordination of the metal (which results in a so-called entatic or rack-induced state) modifies the folding of the metallated Pseudomonas aeruginosa azurin. Our free-energy functional-based approach directly yields the proper nonlinear extra-thermodynamic free energy relationships for the kinetics of folding the wild type and several point-mutated variants of the metallated protein. The results agree quite well with corresponding laboratory experiments. Moreover, our modified free-energy functional provides a sufficient level of detail to explicitly model how the geometric entatic state of the metal modifies the dynamic folding nucleus of azurin.

Streptococcus pneumoniae PstS Production Is Phosphate Responsive and Enhanced during Growth in the Murine Peritoneal Cavity

ABSTRACT Differential display-PCR (DDPCR) was used to identify aStreptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation ofpstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

Solvation of the folding-transition state in Pseudomonas aeruginosa azurin is modulated by metal: Solvation of azurin's folding nucleus.

The role of water in protein folding, specifically its presence or not in the transition‐state structure, is an unsolved question. There are two common classes of folding‐transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (φ) values, and polarized transition states, which instead display distinct substructures with very high φ‐values. Apo‐and zinc‐forms of Pseudomonas aeruginosa azurin both fold in two‐state equilibrium and kinetic reactions; while the apo‐form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding‐transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic‐to‐alanine φ‐value analysis, valine‐to‐threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding‐transition state upon combination with regular φ‐values. We find that the transition state for folding of apo‐azurin appears completely dry, while that for zinc‐azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo‐and holo‐folding nuclei can be rationalized in terms of the shape of the free‐energy barrier.