Structural Insights into the Dynamic Process of ß2-Adrenergic Receptor Signaling.

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the ß2-adrenergic receptor (ß2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound ß2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Utilization of plant extracts to control the safety and quality of fried foods-A review.

Frying is one of the most common methods of preparing foods. However, it may lead to the formation of potentially hazardous substances, such as acrylamide, heterocyclic amines, trans fatty acids, advanced glycation end products, hydroxymethyl furfural and polycyclic aromatic hydrocarbons, and adversely alter the desirable sensory attributes of foods, thereby reducing the safety and quality of fried foods. Currently, the formation of toxic substances is usually reduced by pretreatment of the raw materials, optimization of process parameters, and the use of coatings. However, many of these strategies are not highly effective at inhibiting the formation of these undesirable reaction products. Plant extracts can be used for this purpose because of their abundance, safety, and beneficial functional attributes. In this article, we focus on the potential of using plant extracts to inhibit the formation of hazardous substances, so as to improve the safety of fried food. In addition, we also summarized the effects of plant extracts, which inhibit the production of hazardous substances, on food sensory aspects (flavor, color, texture, and taste). Finally, we highlight areas where further research is required.

Study on the bistable vibration behaviour of a rod-fastened rotor-bearing system.

Gas turbine generator sets are widely used in IGCC system, gas-steam combine cycle, distributed energy system et al. for its advantages of low pollution, high efficiency, quick start and stop. The structure of gas turbine rotor can be divided into integral rotor and rod-fastened rotor. Experimental study shows that the vibration signal, especially the displacement signal, of the rod-fastened rotor will increase/decrease greatly in a small interval of rotating speed. The reason for this phenomenon is the unique structure of the rod-fastened rotor, namely the interfaces between discs. In this paper, based on the Lagrange equation, the equation of motion of a rod-fastened rotor-bearing system considering the damping of the contact interface is established. The bistable behaviour and hysteretic cycle, also called the jumping phenomenon in engineering, are revealed. In addition, a test bench of the rod-fastened rotor-bearing system is built. The bistable behaviour and hysteretic cycle are experimentally proven, and the effect of the eccentric distance of the rotor on the bistable behaviour is experimentally explored.

Oriented Two-Dimensional Covalent Organic Framework Membranes with High Ion Flux and Smart Gating Nanofluidic Transport.

Nanofluidic ion transport holds high promise in bio-sensing and energy conversion applications. However, smart nanofluidic devices with high ion flux and modulable ion transport capabilities remain to be realised. Herein, we demonstrate smart nanofluidic devices based on oriented two-dimensional covalent organic framework (2D COF) membranes with vertically aligned nanochannel arrays that achieved a 2-3 orders of magnitude higher ion flux compared with that of conventional single-channel nanofluidic devices. The surface-charge-governed ion conductance is dominant for electrolyte concentration up to 0.01 M. Moreover, owing to the customisable pH-responsivity of imine and phenol hydroxyl groups, the COF-DT membranes attained an actively modulable ion transport with a high pH-gating on/off ratio of ≈100. The customisable structure and rich chemistry of COF materials will offer a promising platform for manufacturing nanofluidic devices with modifiable ion/molecular transport features.

Single-Pore versus Dual-Pore Bipyridine-Based Covalent-Organic Frameworks: An Insight into the Heterogeneous Catalytic Activity for Selective CH Functionalization.

Exponential growth in the field of covalent-organic frameworks (COFs) is emanating from the direct correlation between designing principles and desired properties. The comparison of catalytic activity between single-pore and dual-pore COFs is of importance to establish structure-function relationship. Herein, the synthesis of imine-linked dual-pore [(BPyDC)]x % -ETTA COFs (x = 0%, 25%, 50%, 75%, 100%) with controllable bipyridine content is fulfilled by three-component condensation of 4,4',4″,4'″-(ethene-1,1,2,2-tetrayl)tetraaniline (ETTA), 4,4'-biphenyldialdehyde, and 2,2'-bipyridyl-5,5'-dialdehyde in different stoichiometric ratio. The strong coordination of bipyridine moieties of [(BPyDC)]x % -ETTA COFs with palladium imparts efficient catalytic active sites for selective functionalization of sp2 CH bond to CX (X = Br, Cl) or CO bonds in good yield. To broaden the scope of regioselective CH functionalization, a wide range of electronically and sterically substituted substrates under optimized catalytic condition are investigated. A comparison of the catalytic activity of palladium decorated dual-pore frameworks with single-pore imine-linked Pd(II) @ Py-2,2'-BPyDC framework  is undertaken. The finding of this work provides a sporadic example of chelation-assisted CH functionalization and disclosed an in-depth comparison of the relationship between superior catalytic activity and core properties of rationally designed imine linked frameworks.

Mono-/Bimetallic Neutral Iridium(III) Complexes Bearing Diketopyrrolopyrrole-Substituted N-Heterocyclic Carbene Ligands: Synthesis and Photophysics.

The synthesis and photophysics (UV-vis absorption, emission, and transient absorption) of four neutral heteroleptic cyclometalated iridium(III) complexes (Ir-1-Ir-4) incorporating thiophene/selenophene-diketopyrrolopyrrole (DPP)-substituted N-heterocyclic carbene (NHC) ancillary ligands are reported. The effects of thiophene versus selenophene substitution on DPP and bis- versus monoiridium(III) complexation on the photophysics of these complexes were systematically investigated via spectroscopic techniques and density functional theory calculations. All complexes exhibited strong vibronically resolved absorption in the regions of 500-700 nm and fluorescence at 600-770 nm, and both are predominantly originated from the DPP-NHC ligand. Complexation induced a pronounced red shift of this low-energy absorption band and the fluorescence band with respect to their corresponding ligands due to the improved planarity and extended π-conjugation in the DPP-NHC ligand. Replacing the thiophene units by selenophenes and/or biscomplexation led to the red-shifted absorption and fluorescence spectra, accompanied by the reduced fluorescence lifetime and quantum yield and enhanced population of the triplet excited states, as reflected by the stronger triplet excited-state absorption and singlet oxygen generation.

Prognostic values of microRNA-130 family expression in patients with cancer: a meta-analysis and database test.

BACKGROUND: Emerging evidence shows that microRNA-130 (miRNA-130) family may be useful as prognostic biomarkers in cancer. However, there is no confirmation in an independent validation study. The aim of this study was to summarize the prognostic value of miRNA-130 family (miRNA-130a and miRNA-130b) for survival in patients with cancer. METHODS: The pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to estimate the association strength between miRNA-130 family expression and prognosis. Kaplan-Meier plotters were used to verify the miRNA-130b expression and overall survival (OS). RESULTS: A total of 2141 patients with OS and 1159 patients with disease-free survival (DFS)/progression-free survival (PFS) were analyzed in evidence synthesis. For the miRNA-130a, the overall pooled effect size (HR) was HR 1.58 (95% CI: 1.21-2.06, P < 0.001). Tissue and serum expression of miRNA-130a was significantly associated with the OS (HR = 1.54, 95% CI: 1.11-2.14, P = 0.009; HR = 1.65, 95% CI: 1.14-2.38, P = 0.008), and in gastric cancer (HR = 1.81, 95% CI: 1.34-2.45, P < 0.001). For the miRNA-13b, a statistical correlation was observed between high miRNA-130b expression and poor OS in patients with cancer (HR = 1.95, 95% CI: 1.47-2.59, P < 0.001), especially in tissue sample (HR = 2.01, 95% CI: 1.39-2.91, P < 0.001), Asian (HR = 2.55, 95% Cl: 1.77-3.69, P < 0.001) and hepatocellular carcinoma (HR = 1.87, 95% CI: 1.23-2.85, P = 0.004). The expression of miRNA-130b was significantly correlated with DFS/PFS (HR = 1.53, 95% CI: 1.31-1.77, P < 0.001), in tissue (HR = 1.98, 95% CI: 1.50-2.62, P < 0.001) and serum (HR = 1.37, 95% CI: 1.15-1.64, P < 0.001), especially in HCC (HR = 1.98, 95% CI: 1.50, 2.62, P < 0.001). In database test, a significant correlation between high miRNA-130b expression and poor OS for HCC patients was observed (HR = 1.55, 95% CI: 1.01, 2.35, P = 0.0045). CONCLUSION: The high expression of miRNA-130 family might predict poor prognosis in cancer patients. Prospectively, combining miRNA-130a and miRNA-130b may be considered as powerful prognostic predictor for clinical application.

How Do Enzymes Orient When Trapped on Metal-Organic Framework (MOF) Surfaces?

Enzyme immobilization in metal-organic frameworks (MOFs) offers retained enzyme integrity and activity, enhanced stability, and reduced leaching. Trapping enzymes on MOF surfaces would allow for catalysis involving large substrates. In both cases, the catalytic efficiency and selectivity depend not only on enzyme integrity/concentration but also orientation. However, it has been a challenge to determine the orientation of enzymes that are supported on solid matrices, which is even more challenging for enzymes immobilized/trapped in MOFs due to the interferences of the MOF background signals. To address such challenge, we demonstrate in this work the utilization of site-directed spin labeling in combination with Electron Paramagnetic Resonance spectroscopy, which allows for the first time the characterization of the orientation of enzymes trapped on MOF surfaces. The obtained insights are fundamentally important for MOF-based enzyme immobilization design and understanding enzyme orientation once trapped in solid matrices or even cellular confinement conditions.

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein-Polymer Hybrid.

Protein-polymer conjugates are attractive biomaterials which combine the functions of both proteins and polymers. The bioactivity of these hybrid materials, however, is often reduced upon conjugation. It is important to determine and monitor the protein structure and active site availability in order to optimize the polymer composition, attachment point, and abundance. The challenges in probing these insights are the large size and high complexity in the conjugates. Herein, we overcome the challenges by combining electron paramagnetic resonance (EPR) spectroscopy and atomic force microscopy (AFM) and characterize the structure of antibacterial hybrids formed by polyethylene glycol (PEG) and an antibacterial protein. We discovered that the primary reasons for activity loss were PEG blocking the substrate access pathway and/or altering protein surface charges. Our data indicated that the polymers tended to stay away from the protein surface and form a coiled conformation. The structural insights are meaningful for and applicable to the rational design of future hybrids.

Biophysical and Biochemical Characterization of Avian Secretory Component Provides Structural Insights into the Evolution of the Polymeric Ig Receptor.

The polymeric Ig receptor (pIgR) transports polymeric Abs across epithelia to the mucosa, where proteolytic cleavage releases the ectodomain (secretory component [SC]) as an integral component of secretory Abs, or as an unliganded protein that can mediate interactions with bacteria. SC is conserved among vertebrates, but domain organization is variable: mammalian SC has five domains (D1-D5), whereas avian, amphibian, and reptilian SC lack the D2 domain, and fish SC lacks domains D2-D4. In this study, we used double electron-electron resonance spectroscopy and surface plasmon resonance binding studies to characterize the structure, dynamics, and ligand binding properties of avian SC, avian SC domain variants, and a human SC (hSC) variant lacking the D2 domain. These experiments demonstrated that, unlike hSC, which adopts a compact or "closed" domain arrangement, unliganded avian SC is flexible and exists in both closed and open states, suggesting that the mammalian SC D2 domain stabilizes the closed conformation observed for hSC D1-D5. Experiments also demonstrated that avian and mammalian pIgR share related, but distinct, mechanisms of ligand binding. Together, our data reveal differences in the molecular recognition mechanisms associated with evolutionary changes in the pIgR protein.

Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure.

Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron-electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼ 3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.

Behaviors and kinetics of toluene adsorption-desorption on activated carbons with varying pore structure.

This work was undertaken to investigate the behaviors and kinetics of toluene adsorption and desorption on activated carbons with varying pore structure. Five kinds of activated carbon from different raw materials were selected. Adsorption isotherms and breakthrough curves for toluene were measured. Langmuir and Freundlich equations were fitted to the equilibrium data, and the Freundlich equation was more suitable for simulating toluene adsorption. The process consisted of monolayer, multilayer and partial active site adsorption types. The effect of the pore structure of the activated carbons on toluene adsorption capacity was investigated. The quasi-first-order model was more suitable for describing the process than the quasi-second-order model. The adsorption data was also modeled by the internal particle diffusion model and it was found that the adsorption process could be divided into three stages. In the external surface adsorption process, the rate depended on the specific surface area. During the particle diffusion stage, pore structure and volume were the main factors affecting adsorption rate. In the final equilibrium stage, the rate was determined by the ratio of meso- and macro-pores to total pore volume. The rate over the whole adsorption process was dominated by the toluene concentration. The desorption behavior of toluene on activated carbons was investigated, and the process was divided into heat and mass transfer parts corresponding to emission and diffusion mechanisms, respectively. Physical adsorption played the main role during the adsorption process.

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron-electron resonance.

The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.

Structural and energetic determinants of adhesive binding specificity in type I cadherins.

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell-cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles.

Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.8 ± 5.0 mL/mol and 1.45 ± 0.33 kbar, respectively, which corresponds to a Keq° of 0.13 ± 0.06, so that only 15% of the enzyme adopts the pressure-promoted conformation at ambient pressure. This pressure-promoted displacement of the equilibrium is eliminated by the addition of testosterone, an allosteric activator. Using site-directed spin labeling, we demonstrated that the pressure- and testosterone-sensitive transition is also revealed by pressure-induced changes in the electron paramagnetic resonance spectra of a nitroxide side chain placed at position 85 or 409 of the enzyme. Furthermore, we observed a pressure-induced displacement of the emission maxima of a solvatochromic fluorophore (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin) placed at the same positions, which suggests a relocation to a more polar environment. Taken together, the results reveal an effector-dependent conformational equilibrium between open and closed states of CYP3A4 that involves a pronounced change at the interface between the region of α-helices A/A' and the meander loop of the enzyme, where residues 85 and 409 are located. Our study demonstrates the high potential of pressure-perturbation strategies for studying protein conformational landscapes.

Conformational Flexibility Enables the Function of a BECN1 Region Essential for Starvation-Mediated Autophagy.

BECN1 is essential for autophagy, a critical eukaryotic cellular homeostasis pathway. Here we delineate a highly conserved BECN1 domain located between previously characterized BH3 and coiled-coil domains and elucidate its structure and role in autophagy. The 2.0 Å sulfur-single-wavelength anomalous dispersion X-ray crystal structure of this domain demonstrates that its N-terminal half is unstructured while its C-terminal half is helical; hence, we name it the flexible helical domain (FHD). Circular dichroism spectroscopy, double electron-electron resonance-electron paramagnetic resonance, and small-angle X-ray scattering (SAXS) analyses confirm that the FHD is partially disordered, even in the context of adjacent BECN1 domains. Molecular dynamic simulations fitted to SAXS data indicate that the FHD transiently samples more helical conformations. FHD helicity increases in 2,2,2-trifluoroethanol, suggesting it may become more helical upon binding. Lastly, cellular studies show that conserved FHD residues are required for starvation-induced autophagy. Thus, the FHD likely undergoes a binding-associated disorder-to-helix transition, and conserved residues critical for this interaction are essential for starvation-induced autophagy.

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants.

The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering.

ESR spectroscopy identifies inhibitory Cu2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity.

The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu(2+) ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu(2+) coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu(2+)-ion based long-range distance constraints. Double electron-electron resonance data yield Cu(2+)-Cu(2+) and Cu(2+)-nitroxide distances that are uniquely consistent with one Cu(2+) bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu(2+) ions bind per complex. Unexpectedly, Mg(2+)-catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu(2+) binding at these hitherto unknown sites, 13 Å away from the Mg(2+) positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu(2+) ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu(2+), the Mg(2+)-dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable double-strand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg(2+) in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg(2+). Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.