Jennifer S. Laurence

The role of thiols and disulfides on protein stability.

There has been a tremendous increase in the number of approved drugs derived from recombinant proteins; however, their development as potential drugs has been hampered by their instability that causes difficulty to formulate them as therapeutic agents. It has been shown that the reactivity of thiol and disulfide functional groups could catalyze chemical (i.e., oxidation and beta-elimination reactions) and physical (i.e., aggregation and precipitation) degradations of proteins. Because most proteins contain a free Cys residue or/and a disulfide bond, this review is focused on their roles in the physical and chemical stability of proteins. The effect of introducing a disulfide bond to improve physical stability of proteins and the mechanisms of degradation of disulfide bond were discussed. The qualitative/quantitative methods to determine the presence of thiol in the Cys residue and various methods to derivatize thiol group for improving protein stability were also illustrated.

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

Background: Steroidogenic cytochrome P450 17A1 (CYP17A1) performs hydroxylase and lyase reactions, with only the latter facilitated by cytochrome b5. Results: NMR mapping confirms the CYP17A1/b5 interface and reveals substrate modulation of the interaction. Conclusion: Allosteric communication exists between the buried CYP17A1 active site and its peripheral b5 binding site. Significance: The CYP17A1 reaction mechanism may be governed by proximal conformational control. The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

High-field solution NMR spectroscopy as a tool for assessing protein interactions with small molecule ligands.

The ability of a small molecule to bind and modify the activity of a protein target at a specific site greatly impacts the success of drugs in the pharmaceutical industry. One of the most important tools for evaluating these interactions has been high-field solution nuclear magnetic resonance (NMR) because of its unique ability to examine even weak protein-drug interactions at high resolution. NMR can be used to evaluate the structural, thermodynamic and kinetic aspects of a binding reaction. The basis of NMR screening experiments is that binding causes a perturbation in the physical properties of both molecules. Unique properties of small and macromolecules allow selective detection of either the protein target or ligand, even in a mixture of compounds. This review outlines current methodologies for assessing protein-ligand interactions from the perspectives of the protein target and ligand and delineates the fundamental principles for understanding NMR approaches in drug research. Advances in instrumentation, pulse sequences, isotopic labeling strategies, and the development of competition experiments support the study of higher molecular weight protein targets, facilitate higher-throughput and expand the range of binding affinities that can be evaluated, enhancing the utility of NMR for identifying and characterizing potential therapeutics to druggable protein targets.

Proteins, Pathogens, and Failure at the Composite-Tooth Interface:

In the United States, composites accounted for nearly 70% of the 173.2 million composite and amalgam restorations placed in 2006 (Kingman et al., 2012), and it is likely that the use of composite will continue to increase as dentists phase out dental amalgam. This trend is not, however, without consequences. The failure rate of composite restorations is double that of amalgam (Ferracane, 2013). Composite restorations accumulate more biofilm, experience more secondary decay, and require more frequent replacement. In vivo biodegradation of the adhesive bond at the composite-tooth interface is a major contributor to the cascade of events leading to restoration failure. Binding by proteins, particularly gp340, from the salivary pellicle leads to biofilm attachment, which accelerates degradation of the interfacial bond and demineralization of the tooth by recruiting the pioneer bacterium Streptococcus mutans to the surface. Bacterial production of lactic acid lowers the pH of the oral microenvironment, erodes hydroxyapatite in enamel and dentin, and promotes hydrolysis of the adhesive. Secreted esterases further hydrolyze the adhesive polymer, exposing the soft underlying collagenous dentinal matrix and allowing further infiltration by the pathogenic biofilm. Manifold approaches are being pursued to increase the longevity of composite dental restorations based on the major contributing factors responsible for degradation. The key material and biological components and the interactions involved in the destructive processes, including recent advances in understanding the structural and molecular basis of biofilm recruitment, are described in this review. Innovative strategies to mitigate these pathogenic effects and slow deterioration are discussed.

Human Cytochrome P450 17A1 Conformational Selection: Modulation by Ligand and Cytochrome b5

Background: Crystallography provides a static structure of cytochrome P450 17A1 (CYP17A1). Results: Solution NMR reveals an ensemble of CYP17A1 conformational substates. Conclusion: Ligand, cytochrome b5, or temperature alters the conformational CYP17A1 substates present. Significance: Changes in conformations probably modulate human steroidogenesis by CYP17A1. Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.

Novel Tripeptide Model of Nickel Superoxide Dismutase

Nickel superoxide dismutase (Ni-SOD) catalyzes the disproportionation of superoxide to molecular oxygen and hydrogen peroxide, but the overall reaction mechanism has yet to be determined. Peptide-based models of the 2N:2S nickel coordination sphere of Ni-SOD have provided some insight into the mechanism of this enzyme. Here we show that the coordination sphere of Ni-SOD can be mimicked using the tripeptide asparagine-cysteine-cysteine (NCC). NCC binds nickel with extremely high affinity at physiological pH with 2N:2S geometry, as demonstrated by electronic absorption and circular dichroism (CD) data. Like Ni-SOD, Ni-NCC has mixed amine/amide ligation that favors metal-based oxidation over ligand-based oxidation. Electronic absorption, CD, and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic Ni(II) center bound in square-planar geometry. Ni-NCC is quasi-reversibly oxidized with a midpoint potential of 0.72(2) V (vs Ag/AgCl) and breaks down superoxide in an enzyme-based assay, supporting its potential use as a model for Ni-SOD chemistry.

MAPping the Chiral Inversion and Structural Transformation of a Metal-Tripeptide Complex Having Ni-Superoxide Dismutase Activity

The metal abstraction peptide (MAP) tag is a tripeptide sequence capable of abstracting a metal ion from a chelator and binding it with extremely high affinity at neutral pH. Initial studies on the nickel-bound form of the complex demonstrate that the tripeptide asparagine-cysteine-cysteine (NCC) binds metal with 2N:2S, square planar geometry and behaves as both a structural and functional mimic of Ni superoxide dismutase (Ni-SOD). Electronic absorption, circular dichroism (CD), and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic Ni(II) center. It is apparent from the CD signal of Ni-NCC that the optical activity of the complex changes over time. Mass spectrometry data show that the mass of the complex is unchanged. Combined with the CD data, this suggests that chiral rearrangement of the complex occurs. Following incubation of the nickel-containing peptide in D(2)O and back-exchange into H(2)O, incorporation of deuterium into non-exchangeable positions is observed, indicating chiral inversion occurs at two of the α carbon atoms in the peptide. Control peptides were used to further characterize the chirality of the final nickel-peptide complex, and density functional theory (DFT) calculations were performed to validate the hypothesized position of the chiral inversions. In total, these data indicate Ni-SOD activity is increased proportionally to the degree of structural change in the complex over time. Specifically, the relationship between the change in CD signal and change in SOD activity is linear.

Quantitative analysis of aqueous phase composition of model dentin adhesives experiencing phase separation

There have been reports of the sensitivity of our current dentin adhesives to excess moisture, for example, water-blisters in adhesives placed on over-wet surfaces, and phase separation with concomitant limited infiltration of the critical dimethacrylate component into the demineralized dentin matrix. To determine quantitatively the hydrophobic/hydrophilic components in the aqueous phase when exposed to over-wet environments, model adhesives were mixed with 16, 33, and 50 wt % water to yield well-separated phases. Based upon high-performance liquid chromatography coupled with photodiode array detection, it was found that the amounts of hydrophobic BisGMA and hydrophobic initiators are less than 0.1 wt % in the aqueous phase. The amount of these compounds decreased with an increase in the initial water content. The major components of the aqueous phase were hydroxyethyl methacrylate (HEMA) and water, and the HEMA content ranged from 18.3 to 14.7 wt %. Different BisGMA homologues and the relative content of these homologues in the aqueous phase have been identified; however, the amount of crosslinkable BisGMA was minimal and, thus, could not help in the formation of a crosslinked polymer network in the aqueous phase. Without the protection afforded by a strong crosslinked network, the poorly photoreactive compounds of this aqueous phase could be leached easily. These results suggest that adhesive formulations should be designed to include hydrophilic multimethacrylate monomers and water compatible initiators.

Enzyme activity of phosphatase of regenerating liver is controlled by the redox environment and its C-terminal residues.

Phosphatase of regenerating liver-1 (PRL-1) belongs to a unique subfamily of protein tyrosine phosphatases (PTPases) associated with oncogenic and metastatic phenotypes. While considerable evidence supports a role for PRL-1 in promoting proliferation, the biological regulators and effectors of PRL-1 activity remain unknown. PRL-1 activity is inhibited by disulfide bond formation at the active site in vitro, suggesting PRL-1 may be susceptible to redox regulation in vivo. Because PRL-1 has been observed to localize to several different subcellular locations and cellular redox conditions vary with tissue type, age, stage of cell cycle, and subcellular location, we determined the reduction potential of the active site disulfide bond that controls phosphatase activity to improve our understanding of the function of PRL-1 in various cellular environments. We used high-resolution solution NMR spectroscopy to measure the potential and found it to be -364.3 +/- 1.5 mV. Because normal cellular environments range from -170 to -320 mV, we concluded that nascent PRL-1 would be primarily oxidized inside cells. Our studies show that a significant conformational change accompanies activation, suggesting a post-translational modification may alter the reduction potential, conferring activity. We further demonstrate that alteration of the C-terminus renders the protein reduced and active in vitro, implying the C-terminus is an important regulator of PRL-1 function. These data provide a basis for understanding how subcellular localization regulates the activity of PRL-1 and, with further investigation, may help reveal how PRL-1 promotes unique outcomes in different cellular systems, including proliferation in both normal and diseased states.

Cytochrome P450 17A1 interactions with the FMN domain of its reductase as characterized by NMR

To accomplish key physiological processes ranging from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase. Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b5, for human steroidogenic CYP17A1, the cytochrome P450 reductase FMN domain delivers both electrons, and b5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein. Formation of the CYP17A1·FMN domain complex induced differential line broadening of the NMR signal for each protein. Alterations in the exchange dynamics generally occurred for residues near the surface of the flavin mononucleotide, including 87–90 (loop 1), and for key CYP17A1 active site residues. These interactions were modulated by the identity of the substrate in the buried CYP17A1 active site and by b5. The FMN domain outcompetes b5 for binding to CYP17A1 in the three-component system. These results and comparison with previous NMR studies of the CYP17A1·b5 complex suggest a model of CYP17A1 enzyme regulation.