Control of thiolate nucleophilicity and specificity in zinc metalloproteins by hydrogen bonding: lessons from model compound studies

A single hydrogen bond between an amide N-H and a thiolate sulfur in model complexes designed to mimic the binding site of zinc thiolate proteins, is shown to reduce the reactivity of the thiolate toward electrophiles by up to 2 orders of magnitude. In addition a single such bond is also sufficient to achieve nearly 100% regiospecificity of reaction between a strong, and hence inherently indiscriminate, alkylating agent like trimethyl oxonium tetrafluoroborate and a single sulfur in a dithiolate construct. The importance of these results in understanding how two systems such as the zinc fingers of the GATA family and the Escherichia coli DNA repair protein Ada which share the same pseudotetrahedral structure and tetrascysteinyl ligation around the zinc can fulfill such widely divergent (structural vs reactive) roles and how specificity of reaction in multithiolate-containing systems can be achieved is discussed.     

H-bonding interactions and control of thiolate nucleophilicity and specificity in model complexes of zinc metalloproteins

It is shown in model complexes designed to mimic the binding site of zinc-thiolate proteins that a single hydrogen bond between an amide N-H and a Zn-coordinated thiolate reduces its reactivity toward electrophiles by up to 2 orders of magnitude. In addition, we show that a single N-H...S hydrogen bond is sufficient to achieve near 100% regiospecificity of reaction between a strong, and hence inherently indiscriminate, alkylating agent like trimethyloxonium tetraflouroborate and a single sulfur in a dithiolate construct. The importance of these results in understanding how systems such as the zinc fingers of the GATA family and the E. coli DNA repair protein Ada, which share the same pseudotetrahedral structure and tetracysteinyl ligation around the zinc, can fulfill such widely divergent (structural vs reactive) roles and how specificity of reaction in such multi-thiolate containing systems can be achieved is discussed.     

Template engineering through epitope recognition: a modular, biomimetic strategy for inorganic nanomaterial synthesis

Natural systems often utilize a single protein to perform multiple functions. Control over functional specificity is achieved through interactions with other proteins at well-defined epitope binding sites to form a variety of functional coassemblies. Inspired by the biological use of epitope recognition to perform diverse yet specific functions, we present a Template Engineering Through Epitope Recognition (TEThER) strategy that takes advantage of noncovalent, molecular recognition to achieve functional versatility from a single protein template. Engineered TEThER peptides span the biologic-inorganic interface and serve as molecular bridges between epitope binding sites on protein templates and selected inorganic materials in a localized, specific, and versatile manner. TEThER peptides are bifunctional sequences designed to noncovalently bind to the protein scaffold and to serve as nucleation sites for inorganic materials. Specifically, we functionalized identical clathrin protein cages through coassembly with designer TEThER peptides to achieve three diverse functions: the bioenabled synthesis of anatase titanium dioxide, cobalt oxide, and gold nanoparticles in aqueous solvents at room temperature and ambient pressure. Compared with previous demonstrations of site-specific inorganic biotemplating, the TEThER strategy relies solely on defined, noncovalent interactions without requiring any genetic or chemical modifications to the biomacromolecular template. Therefore, this general strategy represents a mix-and-match, biomimetic approach that can be broadly applied to other protein templates to achieve versatile and site-specific heteroassemblies of nanoscale biologic-inorganic complexes.     

Site-selective metal binding by designed alpha-helical peptides

It is known that the designed alpha-helical peptide family TRI [(Ac-G(LKALEEK)4G-CONH2)], containing single site substitution of a cysteine for a leucine, is capable of binding Cd(II) within a three-stranded coiled coil. The binding affinity of cadmium is dependent upon the site of substitution, with cysteine incorporated at the a site leading to cadmium complexes of higher affinity than when a d site was modified. In this work we have examined whether this differential binding affinity can be expressed in a di-cysteine-substituted peptide in order to develop site specificity within a designed system. The peptide TRI L9CL19C was used to determine whether significant differences in binding affinities at nearly proximal sites could be achieved in a short designed peptide. On the basis of 113Cd, 1H NMR, and circular dichroic spectroscopies, we have shown that 1 equiv of Cd(II) binds exclusively at the a site. Only after that position is filled does the second site become populated. Thus, the TRI system represents the first example where stoichiometrically equivalent peptides with different sequences form the framework for designing molecular assemblies that show site-specific ion recognition. We propose that the distinct metal affinities are due to the cysteine conformers at different substitution points along the peptide. Furthermore, we have shown that site selectivity in biomolecules can be encoded into relatively short peptides with helical sequences and, therefore, do not necessarily require the extensive protein scaffolds found in natural systems.     

Controlled liquid-solid interactions at the micron-scale: towards self-assembling MEMS

Self-assembly is the spontaneous and reversible organization of components into ordered structures, representing an alternative to the conventional manufacture of systems made of components from milli to nano scales. First commercial applications of self-assembly have appeared in recent years, for example in the fabrication of radio frequency identification (RFID) tags. However, the full impact of this new approach towards heterogeneous system integration will only be realized once self-assembly can be programmed on demand. A key concept is the “programmable surface” – an interface whose properties can be controlled with high spatial and temporal resolution. Several crucial topics are discussed: real time control of interfacial properties; optimization of binding site designs; and algorithms for the modeling and control of self-assembly. Promising novel manufacturing methods are emerging that combine the precision and reproducibility of semiconductor fabrication with the scalability and parallelism of stochastic self-assembly and with the specificity and programmability of biochemical processes.     

Proteomics meets cell biology: the establishment of subcellular proteomes

Proteome research aims to unravel the biological complexity encoded by the genome. Due to the complexity of higher eukaryotic cells, single-step characterization of a proteome is likely to be difficult to achieve. However, advantage can be taken of the macromolecular architecture of a cell, e.g., subcellular compartments, organelles, macromolecular structures and multiprotein complexes, to establish subcellular proteomes. This review highlights recent developments in this area of proteomics, namely the establishment of two-dimensional electrophoresis (2-DE) reference maps of subcellular compartments and organelles as well as the characterization of macromolecular structures and multiprotein complexes using a proteomics approach.     

On-line coupling of hollow fiber membranes with electrospray ionization mass spectrometry for continuous affinity selection, concentration and identification of small-molecule libraries.

Combinatorial chemistry has been widely employed in the pharmaceutical industry in the effort towards drug discovery. Rapid and sensitive screening of lead candidates among library compounds has thus imposed significant analytical challenges in recent years. This work involved the development of a continuous affinity capture and concentration system, providing cost-effective and structural analysis of drug candidates in a flow-through format. The system combines the strengths of a hollow fiber dialysis membrane of ease and speed of purification and concentration with the specificity of affinity interactions in solution. The complexes between the lead compounds and the affinity binding proteins are separated from other chemical components inside a dialysis hollow fiber as the result of their differences in size. The affinity complexes are further concentrated inside a second dialysis fiber. The concentrated drug candidates are liberated from the binding proteins in a microdialysis junction and can be directly identified using electrospray ionization mass spectrometry. Two model systems, including human serum albumin-warfarin-related compounds and anti-phenobarbital antibody-barbiturates, were employed for mechanistic studies of dialysis versus dissociation kinetics and competitive selection of drug candidates according to their binding strengths.     

Detection and characterization of nonspecific,sparsely populated binding modes in the early stages of complexation

A method is proposed to study protein–ligand binding in a system governed by specific and nonspecific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultraweak associations lead instead to broader distributions, a manifestation of nonspecific, sparsely populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first‐encounter modes are metastable states from which stable (prerelaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide–protein complex. The peptide adopts several low‐population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated. © 2015 Wiley Periodicals, Inc.     

Automated molecular simulation based binding affinity calculator for ligand-bound HIV-1 proteases

The successful application of high throughput molecular simulations to determine biochemical properties would be of great importance to the biomedical community if such simulations could be turned around in a clinically relevant timescale. An important example is the determination of antiretroviral inhibitor efficacy against varying strains of HIV through calculation of drug-protein binding affinities. We describe the Binding Affinity Calculator (BAC), a tool for the automated calculation of HIV-1 protease-ligand binding affinities. The tool employs fully atomistic molecular simulations alongside the well established molecular mechanics Poisson-Boltzmann solvent accessible surface area (MMPBSA) free energy methodology to enable the calculation of the binding free energy of several ligand-protease complexes, including all nine FDA approved inhibitors of HIV-1 protease and seven of the natural substrates cleaved by the protease. This enables the efficacy of these inhibitors to be ranked across several mutant strains of the protease relative to the wildtype. BAC is a tool that utilizes the power provided by a computational grid to automate all of the stages required to compute free energies of binding: model preparation, equilibration, simulation, postprocessing, and data-marshaling around the generally widely distributed compute resources utilized. Such automation enables the molecular dynamics methodology to be used in a high throughput manner not achievable by manual methods. This paper describes the architecture and workflow management of BAC and the function of each of its components. Given adequate compute resources, BAC can yield quantitative information regarding drug resistance at the molecular level within 96 h. Such a timescale is of direct clinical relevance and can assist in decision support for the assessment of patient-specific optimal drug treatment and the subsequent response to therapy for any given genotype.     

In‐Situ Spin Labeling of His‐Tagged Proteins: Distance Measurements under In‐Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin‐labeled chemical recognition unit for switchable and concomitantly high affinity binding to His‐tagged proteins was synthesized. In combination with an orthogonal site‐directed spin label, this novel spin probe, Proxyl‐trisNTA (P‐trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate‐binding protein, 2) its substrate‐dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site‐specific spin labeling in cell lysates under in‐cell conditions. This approach will open new avenues towards in‐cell EPR.     

Calculations of interaction energy between certain components of large multimolecular complexes

A scheme is proposed for calculating the energies of interaction between certain components of large multimolecular systems. These energies are used to model the binding and extraction of metal ions and metal complexes in solutions. The procedure can be extended with ease to calculate the energies of interaction between any two components of an arbitrary polyatomic system. Practical application of the scheme is illustrated taking actinide binding by different sorbents in solutions as examples.     

Electrospray ionization mass spectrometry studies of noncovalent myosin VI complexes reveal a new specific calmodulin binding site

Among the myosin superfamily, myosin VI differs from all others by a reverse directionality and a particular motility. Little structural information is available for myosin VI. It is known that it binds one calmodulin (CaM) by means of a single "IQ motif" and that myosin VI contains a specific insert located at the junction between the motor domain (MD) and the lever arm, likely to play a critical role for the unusual motility previously observed. Electrospray ionization mass spectrometry (MS) was used to determine the CaM and Ca2+ stoichiometries in several myosin VI constructs. In particular, the experimental conditions required for the observation of multiprotein/Ca2+ noncovalent assemblies are detailed for two truncated MD constructs (less than 20 kDa) and for three full MD constructs (more than 90 KDa). The specificity of the detected stoichiometries is discussed for each construct and the resolving power of Time of Flight mass spectrometry is stressed, in particular for the detection of metal ions binding to high molecular weight complexes. MS reveals a new CaM binding site for myosin VI and highlights a different behavior for the five myosin VI constructs versus Ca2+ binding. In addition to these stoichiometry based experiments, gas-phase dissociation analyses on intact complexes are described. They reveal that Ca2+ transfer between protein partners occurs during the dissociation process for one construct with a full MD. Charge-transfer and dissociation behavior has allowed to draw structural assumptions for the interaction of the MD with the CaM N-terminal lobe.     

Mass-resolved infrared spectroscopy of complexes without chromophore by nonresonant femtosecond ionization detection

Mass-resolved excitation spectroscopic techniques are usually limited to systems with a chromophore, that is, a functional group with electronic transitions in the Vis/UV, with lifetimes from hundreds of picoseconds to some microseconds. In this paper, we expand such techniques to any system, by using a combination of nanosecond IR pulses with nonresonant ionization with 800 nm femtosecond laser pulses. Furthermore, we demonstrate that the technique can achieve conformational specificity introducing an additional nanosecond IR laser. As a proof-of-principle, we apply the technique to the study of phenol(H(2)O)(1), propofol(H(2)O)(1) γ-butyrolactone(H(2)O)(n), n = 1-3, and (H(2)O)(2) complexes. While monohydrated phenol and propofol clusters permit a direct comparison with a well-studied system including an aromatic chromophore, γ-butyrolactone is a cyclic nonaromatic molecule, whose mass-resolved spectroscopy cannot be tackled by conventional techniques. Finally, we further demonstrate the potential of the technique by obtaining the first mass-resolved IR spectrum of the neutral water dimer, a nice example of a system whose ionization-based detection had not been possible to date.     

Calculation of Host-Guest Binding Affinities Using a Quantum-Mechanical Energy Model

The prediction of protein-ligand binding affinities is of central interest in computer-aided drug discovery, but it is still difficult to achieve a high degree of accuracy. Recent studies suggesting that available force fields may be a key source of error motivate the present study, which reports the first mining minima (M2) binding affinity calculations based on a quantum mechanical energy model, rather than an empirical force field. We apply a semi-empirical quantum-mechanical energy function, PM6-DH+, coupled with the COSMO solvation model, to 29 host-guest systems with a wide range of measured binding affinities. After correction for a systematic error, which appears to derive from the treatment of polar solvation, the computed absolute binding affinities agree well with experimental measurements, with a mean error 1.6 kcal/mol and a correlation coefficient of 0.91. These calculations also delineate the contributions of various energy components, including solute energy, configurational entropy, and solvation free energy, to the binding free energies of these host-guest complexes. Comparison with our previous calculations, which used empirical force fields, point to significant differences in both the energetic and entropic components of the binding free energy. The present study demonstrates successful combination of a quantum mechanical Hamiltonian with the M2 affinity method.     

Study of Cd2+ complexation by the glutathione fragments Cys-Gly (CG) and gamma-Glu-Cys (gamma-EC) by differential pulse polarography

A differential pulse polarographic study of the Cd2+/gamma-Glu-Cys and Cd2+/Cys-Gly systems assisted by the alternating least-squares multivariate curve resolution (MCR-ALS) method was carried out to obtain a better understanding of the different metal affinities of the complexation sites on glutathione (GSH). The simultaneous analysis of the titration of peptide with metal and of metal with peptide allowed the resolution of the Cd2+/Cys-Gly system, whereas in the analysis of the Cd2+/gamma-Glu-Cys system the analysis of a single titration experiment was sufficient. The analysis of the shape of the resulting pure voltammograms and concentration profiles of the resolved components suggested the presence of two different types of bound Cd2+ in the two systems considered, that could be attributed to Cd2+ bound to one or two sulfur atoms to form complexes of stoichiometry 1:1 and 1:2. respectively.     

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

BACKGROUND: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. RESULTS: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. CONCLUSIONS: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.     

Matrix-isolated van der Waals complexes formed between CS2 and dihalogen molecules XY, Where XY = Cl2, Br2, BrCl, ICl, or IBr

Weakly bound 1:1 complexes formed between CS2 and a dihalogen molecule XY = Cl2, Br2, BrCl, ICl, or IBr have each been trapped in an Ar matrix and hence investigated experimentally by their IR spectra as well as theoretically by MP2 and density functional calculations. A planar structure, with an intermolecular angle close to 90 degrees , is expected for such a S=C=S...XY molecular complex. Moreover, for each system involving a heteronuclear dihalogen, two possible complexes exist, viz., S=C=S...XY and S=C=S...YX. The calculated structures, vibrational properties, and binding energetics of the complexes are analyzed, and the NBO formalism is used to interpret their bonding properties. The IR spectra of the complexes thus simulated provided vital guidance for the interpretation of the matrix spectra. For example, complexation was predicted and observed (i) to induce red shifts of the principal absorptions associated with both the CS2 and XY components and (ii) to result, through the change in symmetry, in activation of some modes that are IR-silent for the free components.     

Ion specific surface forces between membrane surfaces

Entities such as ion distributions and forces between lipid membranes depend on effects due to the intervening salt solution that have not been recognized previously. These specific ion or Hofmeister effects influence membrane fusion. A typical illustrative example is this: measurements of forces between double-chained cationic bilayers adsorbed onto molecularly smooth mica surfaces across different 0.6-2 mM salt solutions have revealed a large degree of ion specificity [Pashley et al. J. Phys. Chem. 1986, 90, 1637]. This has been interpreted in terms of very specific anion "binding" to the adsorbed bilayers, as it would too for micelles and other self-assembled systems. However, we show here that inclusion of nonelectrostatic (NES) or ionic dispersion potentials acting between ions and the two surfaces explains such "ion binding". The observed Hofmeister sequence for the calculated pressure without any direct ion binding is given correctly. This demonstrates the importance of a source of ion specificity that has been ignored. It is due to ionic physisorption caused by attractive NES ionic dispersion potentials. There appear to be some far reaching consequences for interpretations of membrane intermolecular interactions in salt solutions.     

Supramolecular assemblies for light-induced electron-transfer reactions

In this paper, we present a summary of our work on highly photostable supramolecular ruthenium complexes, which may be incorporated into more complex systems for artificial solar energy conversion. We have used supramolecular chemistry and photochemistry to synthesize highly photostable ruthenium bipyridine coronates and a bipyridazine podate complex and to enhance photoelectron-transfer reactions in physical model systems for artificial photosynthesis. The recent progress of covalent and non-covalent sensitizer-relay assemblies for highly efficient photoelectron transfer is described.

A detailed mechanistic investigation of the binding behavior of cationic species to crow-ether-modified bipyridine derivatives is presented as an example of supramolecular binding in systems for photoelectron transfer. The host properties of the free ligands and the derived bis-heteroleptic ruthenium complexes are compared using UV—visible, luminescence quenching and proton nuclear magnetic resonance titrations. The combination of these three methods confirms that supramolecular binding of cations and the electron relay methylviologen (MV²⁺) to the complexes can be observed. The binding constants determined are of the order of (1–6) × 10⁴ 1 mol⁻¹ for the crown-ether ruthenium complexes and 1 × 10²−4 × 10³ 1 mol⁻³ for the crown-ether ligands. Single-photon-counting (SPC) investigations give strong indications for the coexistence of different binding mechanisms. The kinetic scheme of Yekta et al. has been adapted to interpret the binding mechanism.     

Thermodynamic basis for promiscuity and selectivity in protein-protein interactions: PDZ domains, a case study

Like other protein-protein interaction domains, PDZ domains are involved in many key cellular processes. These processes often require that specific multiprotein complexes be assembled, a task that PDZ domains accomplish by binding to specific peptide motifs in target proteins. However, a growing number of experimental studies show that PDZ domains (like other protein-protein interaction domains) can engage in a variety of interactions and bind distinct peptide motifs. Such promiscuity in ligand recognition raises intriguing questions about the molecular and thermodynamic mechanisms that can sustain it. To identify possible sources of promiscuity and selectivity underlying PDZ domain interactions, we performed molecular dynamics simulations of 20 to 25 ns on a set of 12 different PDZ domain complexes (for the proteins PSD-95, Syntenin, Erbin, GRIP, NHERF, Inad, Dishevelled, and Shank). The electrostatic, nonpolar, and configurational entropy binding contributions were evaluated using the MM/PBSA method combined with a quasi-harmonic analysis. The results revealed that PDZ domain interactions are characterized by overwhelmingly favorable nonpolar contributions and almost negligible electrostatic components, a mix that may readily sustain promiscuity. In addition, despite the structural similarity in fold and in recognition modes, the entropic and other dynamical aspects of binding were remarkably variable not only across PDZ domains but also for the same PDZ domain bound to distinct ligands. This variability suggests that entropic and dynamical components can play a role in determining selectivity either of PDZ domain interactions with peptide ligands or of PDZ domain complexes with downstream effectors.