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Structural and dynamic behaviors of the green fluorescent protein dimer from jellyfish Clytia gregaria (cgGFP) were investigated by means of molecular dynamics (MD) simulation. Both neutral and ionic forms of the chromophore, p-hydroxybenzylideneimidazolinone (GYS) were considered. The partial atomic charges of the chromophore were derived by BCC and RESP approaches. The structures were compared between the anionic and neutral cgGFP, and between the two subunits (Sub A and Sub B) of the protein dimer. The observed fluorescence intensity and anisotropy decays were further analyzed with theoretical expressions by employing the atomic coordinates of neutral cgGFP obtained by MD simulation. It was assumed that the fluorescence quenching of GYSA and GYSB is ascribed to HB formations between heteroatoms of GYSs and nearby amino acids. Excellent agreement between the observed and calculated intensity decays, and the observed and calculated anisotropy decays were obtained with RESP1 model. The agreements were better in RESP model than those in BCC one. Mean quenching constants of GYSA and GYSB were 0.27 and 0.59 ns-1 overall MD snapshots with RESP1. Mean value of square of direction cosine between the two transition moments of GYSs was 0.74, and that of square of orientation factor was 0.53, and the FRET rates from GYSA to GYSB, and from GYSB to GYSA were 0.87 and 1.87 ns-1.

Water, often termed as the “lubricant of life,” is expected to play an active role in navigating protein dissociation–association reactions. In order to unearth the molecular details, we first compute the free-energy surface (FES) of insulin dimer dissociation employing metadynamics simulation, and then carry out analyses of insulin dimerization and dissociation using atomistic molecular-dynamics simulation in explicit water. We select two sets of initial configurations from 1) the dissociated state and 2) the transition state, and follow time evolution using several long trajectories (∼1–2 μs). During the process we not only monitor configuration of protein monomers, but also the properties of water. Although the equilibrium structural properties of water between the two monomers approach bulklike characteristics at a separation distance of ∼5 nm, the dynamics differ considerably. The complex association process is observed to be accompanied by several structural and dynamical changes of the system, such as large-scale correlated water density fluctuations, coupled conformational fluctuation of protein monomers, a dewettinglike transition with the change of intermonomeric distance R(MM) from ∼4 to ∼2 nm, orientation of monomers and hydrophobic hydration in the monomers. A quasistable, solvent-shared, protein monomer pair (SSPMP) forms at around 2 nm during association process which is a local free-energy minimum having ∼50–60% of native contacts. Simulations starting with arrangements sampled from the transition state (TS) of the dimer dissociation reveal that the final outcome depends on relative orientation of the backbone in the “hotspot” region.

In the course of an earlier investigation into the crystallization of proteins based on the addition of intermolecular ligands, the Kunitz type trypsin inhibitor from soybean (SBTI) was crystallized as a complex with 1,5-disulfonylnaphthalene (ligand library 21D). The two molecules within the asymmetric unit of the monoclinic crystals are related by a near-exact NCS 2-fold axis and have essentially the same conformation as was found for them in previous analyses. The protein dimer is maintained through electrostatic interactions with the sulfonyl groups of four 1,5-disulfonylnaphthalene molecules, which are bound in pairs at the dimer interface and involve symmetrical pairs of arg 30 and arg 47 side chains of each SBTI molecule. The binding of the 1,5-disulfonylnaphthalene molecules at the interface is disordered in that only a coordinated single pair of 1,5-disulfonylnaphthalene molecules can be bound at any one time. There are also molecules of the MES buffer present in the crystal structure that are re...

Human insulin (HI) is an essential protein hormone and its biological activity mostly depends on folded and active conformation in the monomeric state. The present investigation established that Coomassie Brilliant Blue G-250 (CBBG), a small multicyclic hydroxyl compound can reversibly bind to the hormonal protein dimer and maintained most of α-helical folds crucial for biological function of the enzyme. The solution-state 1D NMR and isothermal calorimetric analysis showed a sub-micromolar binding affinity of the molecule to HI. 2D NOESY NMR established that the HI dimer undergoes residue level local conformational change upon binding to CBBG. The chemical shift perturbation and the NOE parameters of active protons of amino acid residues throughout the polypeptides further suggested that CBBG upon binding the protein stabilize α-helixes of both the A and B subunits of the hormonal protein. The changes in Gibb’s free energy (∆G) of the binding was of ~−11.1 kcal/mol and suggested a thermodynamically favourable process. The changes in enthalpy (∆H) and entropy term (T∆S) were −57.2 kcal/mol and −46.1 kcal/mol, respectively. The negative changes in entropy and the NOE transfer effectiveness of several residues in the presence of CBBG molecules indicated that the binding was an enthalpy driven favourable equilibrium process. The NMR-based atomic resolution data and molecular docking studies confirmed that the CBBG binds to HI at the dimeric stage and prevents the availability of the crucial residue segments that partake directly in further oligomerization and subsequent fibrillation. Extended computational analysis based on chemical shift perturbation of protons of active residues further established receptor-ligand based pharmacophore model comprised of 5 hydrophobic and a hydrogen bond acceptor features that can anchor the residues at the A and B chains of HI and inhibit the partial unfolding and hydrophobic collapse to nucleate the fibrillation. Taken together, the results demonstrated that CBBG and their close analogues might be useful to develop a formulation that will maintain the active and functional form of the hormonal protein for a significantly longer time.TOC

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible of a global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting SARS viruses to bypass the host protective response. This work also constitutes a first step of the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.TOC GRAPHICS

Small molecules targeting the PD-1/PD-L1 immune checkpoint are actively searched to offer anticancer oral treatment modalities. Different small molecules have been designed, such as BMS-202 and BMS-1166 which potently bind to PD-L1, sequestering the protein dimer and thus preventing cancer cells to escape antitumor immune responses. A (top → down) deconvolution of BMS compounds has characterized their central biphenyl unit as the minimal element required for PD-L1 protein binding. On this basis, we searched for approved drugs containing a similar biphenyl unit and endowed with immune modulatory activities. We identified the biphenyl anti-inflammatory drug flurbiprofen (FLB) as a potential candidate for PD-L1 interaction, and then proposed a (bottom → up) convolution to select similar molecules, used in Human, susceptible to engage stable interactions with PD-L1. The hypothesis was tested by molecular modeling using the crystal structure of BMS-202 bound to the PD-L1 dimer. The calculations suggest that both (R) and (S) isomers of FLB can form stable complexes with PD-L1, penetrating deep into the cylindric pocket at the interface of the protein dimer. However, the potential energy of interaction (ΔE) is reduced by ~40% for FLB compared to BMS compounds. Then, we identified three FLB analogues (diflunisal, CHF-5074 and HCT1026) forming stable complexes with PD-L1. The longer FLB derivative HCT1026 appears as a suitable binder of the PD-L1 dimer, sliding well along the BMS binding cavity. Our approach proposes a new strategy to discover PD-L1-binding small molecules and raises the intriguing possibility that FLB can bind transiently to PD-L1, thus possibly explaining some of its biological effects. Our study opens new perspectives for the use of FLB (and analogs) as an immune modulator in oncology and other therapeutic domains.

Dimerization of core protein is a crucial step in the formation of the hepatitis C virus (HCV) nucleocapsid, and inhibition of dimer formation is regarded as an attractive approach to design anti-HCV drugs. In this work, we developed the atomic force microscopy based single molecular force spectroscopy (AFM-SMFS) method for the characterization of core protein dimerization with the advantages of small amount of sample consumption and no need of labeling. Interaction force of the core protein with its antibody or aptamer was analyzed to investigate its stoichiometry and binding property. The two specific binding forces were detected due to the probing of dimeric and monomeric core protein, respectively. Moreover, the binding property of protein dimer was different from the monomer. Our work offers a new approach to study the dimerization of core protein, as well as other proteins, and to screen the HCV candidate inhibitors.

The electrostatic (ES) energy of each residue was for the first time quantitatively evaluated in a flavin mononucleotide binding protein (FBP). A residue electrostatic energy (RES) was obtained as the sum of the ES energies between atoms in each residue and all other atoms in the FBP dimer using atomic coordinates obtained by a molecular dynamics (MD) simulation. ES is one of the most important energies among the interaction energies in a protein. It is determined from the RES, the residues which mainly contribute to stabilize the structure of each subunit, and the binding energy between two subunits can be estimated. The RES of all residues in subunit A (Sub A) and subunit B (Sub B) were attractive forces, even though the residues contain net negative or positive charges. This reveals that the ES energies of any of the residues can contribute to stabilize the protein structure. The total binding ES energy over all residues among the subunits was distributed between −0.2 to −1.2 eV (mean = −0.67 eV) from the MD simulation time.

The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments. Human HisGpn1 purity was higher than 95%, molecularly monodisperse and could be concentrated to more than 10 mg/mL without aggregating. Circular dichroism spectra showed that human HisGpn1 was properly folded and displayed a secondary structure rich in alpha helices. HisGpn1 effectively bound GDP and the non-hydrolyzable GTP analogue GMPPCP, and hydrolyzed GTP. We next tested the importance of the C-terminal tail, present in eukaryotic Gpn1 but not in the ancestral archaeal Gpn protein, on HisGpn1 dimer formation. C-terminal deleted human HisGpn1 (HisGpn1ΔC) was also purified as a protein dimer, indicating that the N-terminal GTPase domain contains the interaction surface needed for dimer formation. In contrast to HisGpn1, however, HisGpn1ΔC dimer spontaneously dissociated into monomers. In conclusion, we have developed a method to purify properly folded and functionally active human HisGpn1 from bacteria, and showed that the C-terminal tail, universally conserved in all eukaryotic Gpn1 orthologues, stabilizes the GTPase domain-mediated Gpn1 protein dimer. The availability of recombinant human Gpn1 will open new research avenues to unveil the molecular and pharmacological properties of this essential GTPase.

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. Upon activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 upon stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.

Molecular dynamics of alpha ndash synuclein in the presence of five different salt concentrations show that Zn enhanced the stability of the protein monomer while K shows opposite effect Higher regions of alpha synuclein unfolding was seen between the residues in the presence of NaCl and KCl while the presence of CaCl and MgCl results in similar unfolding between the residues In contrast the unfolding of alpha synuclein in the presence of ZnCl deviate from other salts indicating the influence of Zn in the unfolding of the secondary and tertiary structures of alpha synuclein In protein dimers these misfolding of alpha synuclein are commonly seen between the residues in all these five salts indicating that the NAC region of the dimer is mainly involved in protein unfolding Mutation of Ala Pro clearly shows that decreased the stability of alpha synuclein monomer while Ca influenced protein aggregation in protein dimer Removing the residues from reduced the stability of monomer in the presence of K while shows higher stability with Ca in the protein dimer However both Na and Miens show variations in different forms of the protein These results show that alpha synuclein is less prone to aggregation in the presence of Zn which may have implications for the prevention and therapeutics of the Parkinson rsquo s disease

This article describes the formation of homodimers from their constituting monomers, based on the rules set by a simple model of electric and hydrophobic interactions. These interactions are described in terms of the electric dipole moment (D) and hydrophobic moment vectors (H) of proteins. The distribution of angles formed by the two dipole moments of monomers constituting dimers were analysed, as well as the distribution of angles formed by the two hydrophobic moments. When these distributions were fitted to Gaussian curves, it was found that for biological dimers, the D vectors tend mostly to adopt a perpendicular arrangement with respect to each other, in which the constituting dipoles have the least interaction. A minor population tends towards an antiparallel arrangement implying maximum electric attraction. Also in biological dimers, the H vectors of most monomers tend to interact in such a way that the total hydrophobic moment of the dimer increases with respect to those of the monomers. This shows that hydrophobic moments have a tendency to align. In dimers originating in the crystallisation process, the distribution of angles formed by both hydrophobic and electric dipole moments appeared rather featureless, probably because of unspecific interactions in the crystallisation processes. The model does not describe direct interactions between H and D vectors although the distribution of angles formed by both vectors in dimers was analysed. It was found that in most cases these angles tended to be either small (both moments aligned parallel to each other) or large (antiparallel disposition).

Protein analysis using solid-state nanopores is challenging due to limitations in bandwidth and signal-to-noise ratio. Recent improvements of those two aspects have made feasible the study of small peptides using solid-state nanopores, which have an advantage over biological counterparts in tunability of the pore diameter. Here, we report on the detection and characterization of peptides as small as 33 amino acids. Silicon nitride nanopores with thicknesses less than 10 nm are used to provide signal-to-noise (S/N) levels up to S/N ∼ 10 at 100 kHz. We demonstrate differentiation of monomer and dimer forms of the GCN4-p1 leucine zipper, a coiled-coil structure well studied in molecular biology, and compare with the unstructured 33-residue monomer. GCN4-p1 is sequence segment associated with homodimerization of the transcription factor General Control Nonderepressible 4 (GCN4), which is involved in the control of amino acid synthesis in yeast. The differentiation between two oligomeric forms demonstrates the capabilities of improved solid-state nanopore platforms to extract structural information involving short peptide structures.

Protein aggregation including the formation of dimers and multimers in solution, underlies an array of human diseases such as systemic amyloidosis which is a fatal disease caused by misfolding of native globular proteins damaging the structure and function of affected organs. Different kind of interactors can interfere with the formation of protein dimers and multimers in solution. A very special class of interactors are nanoparticles thanks to the extremely efficient extension of their interaction surface. In particular citrate-coated gold nanoparticles (cit-AuNPs) were recently investigated with amyloidogenic protein $\beta$2-microglobulin ($\beta$2m). Here we present the computational studies on two challenging models known for their enhanced amyloidogenic propensity, namely $\Delta$N6 and D76N $\beta$2m naturally occurring variants, and disclose the role of cit-AuNPs on their fibrillogenesis. The proposed interaction mechanism lies in the interference of the cit-AuNPs with the protein dimers at the early stages of aggregation, that induces dimer disassembling. As a consequence, natural fibril formation can be inhibited. Relying on the comparison between atomistic simulations at multiple levels (enhanced sampling molecular dynamics and Brownian dynamics) and protein structural characterisation by NMR, we demonstrate that the cit-AuNPs interactors are able to inhibit protein dimer assembling. As a consequence, the natural fibril formation is also inhibited, as found in experiment., Comment: Published by RSC, under a Creative Commons Attribution 3.0 Unported Licence

Motivation Accurate molecular structure of the protein dimer representing the elementary building block of intermediate filaments (IFs) is essential towards the understanding of the filament assembly, rationalizing their mechanical properties and explaining the effect of disease-related IF mutations. The dimer contains a ∼300-residue long α-helical coiled coil which cannot be assessed by either direct experimental structure determination or modelling using standard approaches. At the same time, coiled coils are well-represented in structural databases. Results Here we present CCFold, a generally applicable threading-based algorithm which produces coiled-coil models from protein sequence only. The algorithm is based on a statistical analysis of experimentally determined structures and can handle any hydrophobic repeat patterns in addition to the most common heptads. We demonstrate that CCFold outperforms general-purpose computational folding in terms of accuracy, while being faster by orders of magnitude. By combining the CCFold algorithm and Rosetta folding we generate representative dimer models for all IF protein classes. Availability and implementation The source code is freely available at https://github.com/biocryst/IF; a web server to run the program is at http://pharm.kuleuven.be/Biocrystallography/cc. Supplementary information Supplementary data are available at Bioinformatics online.

An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface-enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter-labeled gold nanoparticles (AuNP). Site-specificity is achieved by complexation of nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) anchors on the particle surface by multiple histidines (His6-tag) attached to each C-terminus of the centrosymmetric protein dimer. Correlative single-particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS-inactive AuNP-protein monomers can be directly discriminated from the SERS-active AuNP-protein dimers/oligomers. This information, i.e. the percentage of SERS-active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single-particle approach paves the way for quantitative site-specific SERS assays in which site-specific protein recognition by small chemical and in particular supramolecular ligands can be tested.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson&rsquo, s and Alzheimer&acute, s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.

Refolding is often the bottle-neck step in producing recombinant proteins from inclusion bodies of Escherichia coli, especially for dimer proteins. The refolding process is protein specific, engaging a lot of time and cost to optimize conditions so that the thermodynamics favor protein refolding over competitive aggregation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic agent having significant applications in bone regeneration therapy. In this study, we present a novel solid-phase refolding method for rapid and efficient refolding of recombinant BMP-2 dimer from E. coli. We employed a weak cation exchange resin as the adsorbing support, with decreasing gradient of denaturing agent and exposure to oxidizing conditions for adequate disulfide bond formation. Refolded BMP-2 was further purified using size exclusion chromatography and analyzed for its secondary structure and biological activity. The purified BMP-2 dimer showed dose-dependent induction of alkaline phosphatase (ALP) activity in MC3T3 pre-osteoblast cells, thus translating the success of our refolding method. This simple and rapid method can also be applied in refolding and purification of other BMP-2 like dimer proteins.

The dimeric UVR8 protein is an ultraviolet-B radiation (280-315 nm) photoreceptor responsible for the first step in UV-B regulation of gene expression in plants. Its action comprises the actual absorption of the UV quanta by a tryptophan array at the protein-protein interface, followed by monomerization and subsequent aggregation with downstream signaling components. A crystal structure of the Arabidopsis thaliana tryptophan-rich wild type UVR8 protein dimer was recently published, showing the presence of several salt bridges involving arginines R146, R286, R338, and R354. In this work, molecular dynamics simulations in conjunction with umbrella sampling were used to calculate the binding free energy for the wild type UVR8 dimer and three of its mutants (R286A, R338A, and R286A/R338A), in order to verify whether the key mutants are able to disrupt the dimeric structure as indicated experimentally.

The effects of small (~102 Da) and larger (>103 Da) cosolutes on the equilibrium stability of monomeric globular proteins are broadly understood, excluding volume stabilizes proteins and chemical interactions are stabilizing when repulsive, but destabilizing when attractive. Proteins, however, rarely work alone. Here, we investigate the effects of small and large cosolutes on the equilibrium stability of the simplest defined protein–protein interactions, the side-by-side homodimer formed by the A34F variant of the 56-residue B1 domain of protein G. We used 19F nuclear magnetic resonance spectroscopy to quantify the effects of urea, trimethylamine oxide, Ficoll, and more physiologically relevant cosolutes on the dimer dissociation constant. The data reveal the same stabilizing and destabilizing influences from chemical interactions as observed in studies of protein stability. Results with more physiologically relevant molecules such as bovine serum albumin, lysozyme, and reconstituted Escherichia coli cytosol reflect the importance of chemical interactions between these cosolutes and the test protein. Our study serves as a stepping-stone to a more complete understanding of crowding effects on protein–protein interactions.

We investigated the light-induced crystallization mechanism of proteins. The photochemically produced protein dimer was thought to grow to a crystal size. This was proven experimentally in this study.

Glycobiology, 27 (5), ISSN:0959-6658

A free Cys-SH-containing protein, beta-lactoglobulin (beta-LG), and another protein not possessing the same, viz., apo-alpha-lactoglobulin (apo-alpha-LA), were used in studies to demonstrate the role of this amino acid, along with its secondary structure, in the formation of a protein dimer and a protein-inorganic hybrid nanoflower and in the creation of the peroxidase-like activity of the nanomaterials produced when the proteins were treated with varying Cu2+ concentration under different pH conditions. An increase in the pH as well as the Cu2+ mole ratio results in increasing dimer formation in case of beta-LG due to the presence of free Cys121-SH, while the dimer is not formed in case of apo-alpha-LA under the same conditions. The role of Cys in the dimer formation has been demonstrated both by MALDI and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies. Both of the proteins exhibited changes in their secondary structures to different extents as a function of pH, and the structures were stabilized by Cu2+ interactions, as studied by CD and fluorescence spectroscopy. The small and spherical nanoparticles formed at pH 7 with lower equivalents of Cu2+ join together to form larger aggregates at higher equivalents of Cu2+. For the same concentration at pH 9, both the aggregates and the nanoflowers were noticed. However, at pH 12, the Cu2+ binding induces the formation of fibers along with the flowers. Both the nanoflowers and nanofibers exhibited peroxidase-like activity in a catalytic manner. Nanoflowers were also shown to detect phenol in the concentration range from 10 to 200 mu M. The copper-induced nanobiomaterial obtained in the case of apo-alpha-LA also exhibited peroxidase-like activity. Thus, this paper deals with the green synthesis of copper-induced protein (beta-LG/apo-alpha-LA)-inorganic hybrid nanomaterials that are important due to their applications as nanobiomaterials.

Although the development of chemically induced, self-assembled protein-based materials is rapidly expanding, methods for directing their assembly in solution are sparse, and problems of population heterogeneity remain. By exerting control over the assembly of advanced protein structures, new classes of ordered protein nanomaterials become feasible, affecting numerous applications ranging from therapeutics to nanostructural engineering. Focusing on a protein-based method for modulating the stability of a chemically induced dihydrofolate reductase (DHFR) dimer, we demonstrate the sensitivity of a methotrexate competition assay in determining the change in DHFR–DHFR binding cooperativity via interfacial mutations over a 1.3 kcal/mol range. This represents a change of more than 40% of the dimer complex binding energy conferred from protein–protein cooperativity (~3.1 kcal/mol). With the development of this investigative system and refinement of protein-based techniques for complex stability modulation, the directed assembly of protein nanomaterials into heterocomplexes and a concomitant decrease in population heterogeneity becomes a realizable goal. Copyright © 2012 John Wiley & Sons, Ltd.

The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, α12/13, as a new interaction site for self-assembly. α12/13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two α12/13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two α12/13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 μm. We suggest GTP hydrolysis-driven release of the α12/13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments. Structured digital abstract • hGBP1 and hGBP1 bind by cross-linking study (View interaction) • hGBP1 and hGBP1 bind by molecular sieving (View Interaction: 1, 2) • hGBP1 physically interacts with hGBP1 by two hybrid (View Interaction: 1, 2, 3)

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.

Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide-protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of approximately 53 s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30 cm long. When using Fyn-SH2-SH3 tagged with glutathione S-transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7 s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0+/-2.7 and 58.8+/-6.1 s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that although all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions.

The three dimensional structure of Ferric uptake regulation protein dimer from E. coli, determined by molecular modeling, was docked on a DNA fragment (iron box) and Zn(2+) ions were added in two steps. The first step involved the binding of one Zn2+ ion to what is known as the zinc site which consists of the residues Cys 92, Cys 95, Asp 137, Asp141, Arg139, Glu 140, His 145 and His 143 with an average metal-Nitrogen distance of 2.5 A and metal-oxygen distance of 3.1-3.2 A. The second Zn2+ ion is bound to the iron activating site formed from the residues Ile 50, His 71, Asn 72, Gly 97, Asp 105 and Ala 109. The binding of the second Zn2+ ion strengthened the binding of the first ion as indicated by the shortening of the zinc-residue distances. Fe2+, when added to the complex consisting of 2Zn2+/Fur dimer/DNA, replaced the Zn2+ ion in the zinc site and when a second Fe2+ was added, it replaced the second zinc ion in the iron activating site. The binding of both zinc and iron ions induced a similar change in Fur conformations, but shifted residues closer to DNA in a different manner. This is discussed along with a possible role for the Zn2+ ion in the Fur dimer binding of DNA in its repressor activity.

Analysis of structure and features of subunit contact areas of HIV reverse transcriptase (RT) was done. The amino acid residues from contact areas of RT subunits are more conservative than the other residues of this protein. Estimation of contribution of the amino acid residues from the contact areas into the binding energy of subunits showed that the binding energy is mostly localized on the several amino acid residues of the dimer interface (hot spots). The majority of these hot spots form several clusters on the contact surfaces. Analysis of these clusters allows prediction of the region on the contact area of the protein dimer that can be useful for design RT HIV inhibitors of dimerization.

A disease that caused by dengue virus (DENV) has become the major health problem of the world. Nowadays, no effective treatment is available to overcome the disease due to the level of dengue virus pathogeneses. A novel treatment method such as antiviral drug is highly necessary for coping with the dengue disease. Envelope protein is one of the non-structural proteins of DENV, which engaged in the viral fusion process. The fusion process is mediated by the conformational change in the protein structure from dimer to trimer state. The previous research showed the existing cavity on the dimer structure of the envelope protein. The existing ligand could get into cavity of the envelope protein, stabilize the dimer structure or hamper the transition of dimer protein into trimer. In this fashion, the fusion process can be prevented. The aim of this research is designing the cyclic peptide with prolin-prolin bond as fusion inhibitor of DENV envelope protein through molecular docking and molecular dynamics simulation. The screening of 3,883 cyclic peptides, each of them connected by prolin-prolin bond, through molecular docking resulted in five best ligands. The pharmacological and toxicity character of these five ligands were analised in silico. The result showed that PYRRP was the best ligand. PAWRP was also chosen as the best ligand because it showed good affinity with protein cavity. Stability of ligand-protein complex was analyzed by molecular dynamics simulation. The result showed that PYRRP ligand was able to support the stability of DENV envelope protein dimer structure at 310 K and 312 K. While PAWRP ligand actively formed complex with the DENV envelope protein at 310 K compared to 312 K. Thus the PYRRP ligand has a potential to be developed as DENV fusion inhibitor., 15 pages, ICBS BIO-UGM, Yogyakarta, Indonesia, 20-21 September 2015

This study reports the application of electrospray ionization (ESI) mass spectrometry (MS) with on-line rapid mixing for millisecond time-resolved studies of the refolding and assembly of a dimeric protein complex. Acid denaturation of S100A11 disrupts the native homodimeric protein structure. Circular dichroism and HSQC nuclear magnetic resonance measurements reveal that the monomeric subunits unfold to a moderate degree but retain a significant helicity and some tertiary structural elements. Following a rapid change in solution conditions to a slightly basic pH, the native protein reassembles with an effective rate constant of 6 s(-)(1). The ESI charge state distributions measured during the reaction suggest the presence of three kinetic species, namely, a relatively unfolded monomer (M(U)), a more tightly folded monomeric reaction intermediate (M(F)), and dimeric S100A11. These three forms exhibit distinct calcium binding properties, with very low metal loading levels for M(U), up to two calcium ions for M(F), and up to four for the dimer. Surprisingly, on-line pulsed hydrogen-deuterium exchange (HDX) reveals that each of the monomeric forms of the protein comprises two subspecies that can be distinguished on the basis of their isotope exchange levels. As the reaction proceeds, the more extensively labeled species are depleted. The exponential nature of the measured intensity-time profiles implies that the rate-determining step of the overall process is a unimolecular event. The kinetics are consistent with a sequential folding and assembly mechanism involving two increasingly nativelike monomeric intermediates en route to the native S100A11 dimer.

The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to the heavy metals Cd(II), Zn(II), and Pb(II). Expression of this heavy-metal efflux pump is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. Here we report the X-ray crystal structure of CadC at 1.9 Å resolution. The dimensions of the protein dimer are approximately 30 Å by 40 Å by 70 Å. Each monomer contains six α-helices and a three-stranded β-sheet. Helices 4 and 5 form a classic helix-turn-helix motif that is the putative DNA binding region. The α1 helix of one monomer crosses the dimer to approach the α4 helix of the other monomer, consistent with the previous proposal that these two regulatory metal binding sites for the inducer cadmium or lead are each formed by Cys-7 and Cys-11 from the N terminus of one monomer and Cys-58 and Cys-60 of the other monomer. Two nonregulatory metal binding sites containing zinc are formed between the two antiparallel α6 helices at the dimerization interface. This is the first reported three-dimensional structure of a member of the ArsR/SmtB family with regulatory metal binding sites at the DNA binding domain and the first structure of a transcription repressor that responds to the heavy metals Cd(II) and Pb(II).

Designing proteins with novel protein/protein binding properties can be achieved by combining the tools that have been developed independently for protein docking and protein design. We describe here the sequence-independent generation of protein dimer orientations by protein docking for use as scaffolds in protein sequence design algorithms. To dock monomers into sequence-independent dimer conformations, we use a reduced representation in which the side chains are approximated by spheres with atomic radii derived from known C2 symmetry-related homodimers. The interfaces of C2-related homodimers are usually more hydrophobic and protein core-like than the interfaces of heterodimers; we parameterize the radii for docking against this feature to capture and recreate the spatial characteristics of a hydrophobic interface. A fast Fourier transform-based geometric recognition algorithm is used for docking the reduced representation protein models. The resulting docking algorithm successfully predicted the wild-type homodimer orientations in 65 out of 121 dimer test cases. The success rate increases to approximately 70% for the subset of molecules with large surface area burial in the interface relative to their chain length. Forty-five of the predictions exhibited less than 1 A C(alpha) RMSD compared to the native X-ray structures. The reduced protein representation therefore appears to be a reasonable approximation and can be used to position protein backbones in plausible orientations for homodimer design.

Axonemal and cytoplasmic dyneins share a highly conserved 8 kDa light chain (DLC8) for motor assembly and function. Other than serving as a light chain of dynein complexes, DLC8 has been shown to bind a larger number of proteins with diverse biological functions including cell cycle control, apoptosis, and cell polarity maintenance. Therefore, DLC8 is likely a multifunctional regulatory protein. DLC8 exists as a dimer in solution, and the protein dimer is capable of binding to two target molecules. In this work, the backbone dynamics of DLC8, both in its apo- and target-peptide bound forms, were characterized by 15N NMR relaxation studies. The relaxation data were analyzed using model-free approach. We show that the target peptide-binding region of apo-DLC8 experiences microsecond-to-millisecond time scale conformational fluctuation, suggesting that the target-binding region of the protein is capable of adjusting its shape and size in responding to its various targets. The conformational breathing of the target-binding region of apo-DLC8 was also supported by backbone amide exchange experiment. Such segmental conformational motion of the protein is significantly reduced upon forming a complex with a target peptide. The dynamic properties of DLC8 in solution provide insight into the protein's diverse sequence-dependent target binding.

We demonstrate a novel methodology to disrupt the symmetry in the NMR spectra of homodimers. A paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on only one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. This methodology can be widely used for solution structure determination of symmetric dimers.

Cathepsin K is the major collagenolytic protease in bone that facilitates physiological as well as pathological bone degradation. Despite its key role in bone remodeling and for being a highly sought-after drug target for the treatment of osteoporosis, the mechanism of collagen fiber degradation by cathepsin K remained elusive. Here, we report the structure of a collagenolytically active cathepsin K protein dimer. Cathepsin K is organized into elongated C-shaped protease dimers that reveal a putative collagen-binding interface aided by glycosaminoglycans. Molecular modeling of collagen binding to the dimer indicates the participation of nonactive site amino acid residues, Q21 and Q92, in collagen unfolding. Mutations at these sites as well as perturbation of the dimer protein–protein interface completely inhibit cathepsin-K–mediated fiber degradation without affecting the hydrolysis of gelatin or synthetic peptide. Using scanning electron microscopy, we demonstrate the specific binding of cathepsin K at the edge of the fibrillar gap region of collagen fibers, which suggest initial cleavage events at the N - and C -terminal ends of tropocollagen molecules. Edman degradation analysis of collagen fiber degradation products revealed those initial cleavage sites. We propose that one cathepsin K molecule binds to collagen-bound glycosaminoglycans at the gap region and recruits a second protease molecule that provides an unfolding and cleavage mechanism for triple helical collagen. Removal of collagen-associated glycosaminoglycans prevents cathepsin K binding and subsequently fiber hydrolysis. Cathepsin K dimer and glycosaminoglycan binding sites represent novel targeting sites for the development of nonactive site-directed second-generation inhibitors of this important drug target.

Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 A apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-exposed site (Trp167), time-dependent Stokes shifts and collisional quenching data allow an analysis of intra-protein dynamical communication. A double mutant, Y25A:V260A, was also inserted into each single-Trp construct and analyzed accordingly. None of the mutations affect fluorescence lifetimes, Stokes shift relaxation rates, and quenching data for the surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent probes of the active-site tryptophan 87 reveal distinctive forms of dynamical communication. Stokes shifts show that the distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process not previously reported by such measurements, and the double mutant (Y25A:V260A) eliminates the temperature-dependent transition sensed by the active-site tryptophan in the presence of V260A. Collisional quenching data at Trp87 further show a structural change in the active-site environment/solvation for V260A. In the aggregate, the temperature dependencies of the fluorescence data are distinct from the breaks in behavior previously reported for catalysis and hydrogen/deuterium exchange, attributed to time scales for the interconversion of protein conformational substates that are slower and more global than the local motions monitored within. An extended network of dynamical communication between the protein dimer surface and substrate- and cofactor-binding domains emerges from the flourescent data.

Cation- and anion-exchange chromatography can be used to purify a polyethylene glycol-linked protein dimer (PEG dimer) made with M, 20 000 PEG bis-vinylsulfone, even when there are no net charge differences between the components that are being separated. The retention time on ion-exchange generally is inversely proportional to the PEG:protein ratio (on a mass basis). One of the biggest challenges in developing the process for making this PEG dimer was the quality of the PEG linker. Reversed-phase HPLC can be used to determine both size heterogeneity and the degree of end-group activation of Mr 20 000 PEG bis-vinylsulfone. In addition, we have found that hydrophobic interaction chromatography can be used make more size homogeneous preparations of Mr 20000 PEG bis-vinylsulfone, which significantly increased the recovery of the PEG dimer.

Studies of the stability of HLA-DQ have revealed a correlation between SDS stability of MHC class II αβ dimers and insulin-dependent diabetes mellitus (IDDM) susceptibility. The MHC class II αβ dimer encoded by HLA-DQA1*0102/DQB1*0602 (DQ0602), which is a dominant protective allele in IDDM, exhibits the greatest SDS stability among HLA-DQ molecules in EBV-transformed B-lymphoblastoid cells and PBLs. DQ0602 is also uniquely SDS stable in the HLA-DM-deficient cell line, BLS-1. We addressed the molecular mechanism of the stability of DQ0602 in BLS-1. A panel of mutants based on the polymorphic differences between HLA-DQA1*0102/DQB1*0602 and HLA-DQA1*0102/DQB1*0604 were generated and expressed in BLS-1. An Asp at β57 was found to be critical for SDS stability, whereas Tyr at β30, Gly at β70, and Ala at β86 played secondary roles. Furthermore, the level of class II-associated invariant chain peptide bound to HLA-DQ did not correlate with SDS stability, suggesting that class II-associated invariant chain peptide does not play a direct role in the unique SDS stability of DQ0602. These results support a role for DQB1 codon 57 in HLA-DQ αβ dimer stability and IDDM susceptibility.

The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1–55, named IN1−55) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739–746], consists of four α-helices and is well defined. Helices α1, α2 and α3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix α3. The orientation of the two monomeric units with respect to each other shows considerable variation. 15N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN1−55 shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN1−55 [Cai et al. (1997) Nat. Struct. Biol., 4, 567–577], where the corresponding residues are positively charged and neutral ones.

Using a corrected molar extinction coefficient for yeast apo copper-zinc superoxide dismutase (CuZnSOD), we have confirmed that the metal binding properties of this protein in vitro differ greatly from those of the bovine and human CuZnSOD enzymes. Thus yeast apo CuZnSOD was found to bind only one Co2+ per protein dimer under the conditions in which the bovine and human CuZnSOD apoenzymes readily bind two per dimer. The spectroscopic properties characteristic of the two Cu2+ plus two Co2+ per dimer or four Cu2+ per dimer metal-substituted bovine apo CuZnSOD derivatives were obtained for the yeast apoprotein but by the addition of only half of the appropriate metals, i.e., one Cu2+ plus one Co2+ per dimer or two Cu2+ per dimer. This half-metallated yeast CuZnSOD has been characterized by UV-visible and EPR spectroscopy as well as by native polyacrylamide gel electrophoresis. We conclude that yeast apo CuZnSOD, unlike the bovine and human apoproteins, cannot be reconstituted fully with metal ions under the same conditions. Instead, only one subunit of the homodimer, the "normal" subunit, can be remetalled in a fashion reminiscent of the well-characterized bovine protein. The other "phantom" subunit is not competent to bind metals in this fashion. Furthermore, we have shown that CuZnSOD protein isolated from Saccharomyces cerevisiae that lacks the gene coding for the copper chaperone, Lys7p, contains only one metal ion, Zn2+, per protein dimer. The possibility that yeast CuZnSOD can exist in multiple conformational states may represent an increased propensity of the yeast protein to undergo changes that can occur in all CuZnSODs, and may have implications for amyotrophic lateral sclerosis.

How protein–protein interaction affects protein–nanoparticle self-assembly is the key to the understanding of biomolecular coating of nanoparticle in biological fluids. However, the relationship between protein shape and its interaction with nanoparticles is still under-exploited because of lack of a well-conceived binding system and a method to detect the subtle change in the protein–nanoparticle assemblies. Noticing this unresolved need, we cloned and expressed a His-tagged SpeA protein that adopts a bridge-shaped dimer structure, and utilized a high-resolution capillary electrophoresis method to monitor assembly formation between the protein and quantum dots (QDs, 5 nm in diameter). We observed that the bridge-shaped structure rendered a low SpeA:QD stoichiometry at saturation. Also, close monitoring of imidazole (Im) displacement of surface-bound protein revealed a unique two-step process. High-concentration Im could displace surface-bound SpeA protein and form a transient QD–protein intermediate, through a kinetically controlled displacement process. An affinity-driven equilibrium step then followed, resulting in re-assembling of the QD–protein complex in about 1 h. Through a temporarily formed intermediate, Im causes a rearrangement of His-tagged proteins on the surface. Thus, our work showcases that the synergistic interplay between QD–His-tag interaction and protein–protein interaction can result in unique properties of protein–nanoparticle assembly for the first time.

Griffithsin (Grft) is a protein lectin derived from red algae that tightly binds the HIV envelope protein gp120 and effectively inhibits virus infection. This inhibition is due to the binding by Grft of high-mannose saccharides on the surface of gp120. Grft has been shown to be a tight dimer, but the role of the dimer in Grft's anti-HIV function has not been fully explored. To investigate the role of the Grft dimer in anti-HIV function, an obligate dimer of Grft was designed by expressing the protein with a peptide linker between the two subunits. This "Grft-linker-Grft" is a folded protein dimer, apparently nearly identical in structural properties to the wild-type protein. A "one-armed" obligate dimer was also designed (Grft-linker-Grft OneArm), with each of the three carbohydrate binding sites of one subunit mutated while the other subunit remained intact. While both constructed dimers retained the ability to bind gp120 and the viral surface, Grft-linker-Grft OneArm was 84- to 1,010-fold less able to inhibit HIV than wild-type Grft, while Grft-linker-Grft had near-wild-type antiviral potency. Furthermore, while the wild-type protein demonstrated the ability to alter the structure of gp120 by exposing the CD4 binding site, Grft-linker-Grft OneArm largely lost this ability. In experiments to investigate gp120 shedding, it was found that Grft has different effects on gp120 shedding for strains from subtype B and subtype C, and this might correlate with Grft function. Evidence is provided that the dimer form of Grft is critical to the function of this protein in HIV inhibition. ispartof: Antimicrobial Agents and Chemotherapy vol:57 issue:8 pages:3976-3989 ispartof: location:United States status: published

Gene V protein of bacteriophage f1 is a dimeric protein that binds cooperatively to single-stranded nucleic acids. In order to determine whether a monomer-dimer equilibrium has an appreciable effect upon the thermodynamics of gene V protein binding to nucleic acids, the dissociation constant for the protein dimer was investigated using size-exclusion chromatography. At concentrations ranging from 5 x 10(-10) to 1.2 x 10(-5) M, the Stokes radius of the protein was that expected of the dimer of the gene V protein. The Stokes radius of the protein was also independent of salt concentration from 0.2 to 1.0 M NaCl in a buffer containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The binding of the dimeric gene V protein to poly(dA) was studied using a simplified lattice model for protein-protein interactions adapted for use with a dimeric protein that binds simultaneously to two strands of nucleic acid. Interpretation of the salt dependence, C = [d log(Kint omega)]/[d log(NaCl)], of binding of such a dimeric protein to nucleic acid using the theory of Record et al. (Record, M. T., et al. (1976) J. Mol. Biol. 107, 145-158) indicates that C is a function of the numbers of cations and anions released from protein and nucleic acid upon binding of the dimer, not of the monomer. Cooperativity of gene V protein binding to poly(dA) was studied with titration experiments that are sensitive to the degree of cooperativity of binding. The cooperativity factor omega, defined as the ratio of the binding constant for a site adjacent to a previously bound dimer to that for an isolated site, was found to be relatively insensitive to salt, with a value in the range of 2000-7000 for binding to poly(dA) at 3 degrees C and at 23 degrees C. This high cooperativity factor supports the suggestion that protein-protein contacts play a major role in the formation of the superhelical gene V protein-single-stranded nucleic acid complex.

Chemical cross-linking has been used to determine the composition of the erythrocyte band 3 protein dimer in Southeast Asian ovalocytes (SAO). Individuals with SAO are heterozygous for a mutation in which residues 400-408 of band 3 are deleted. Normal and variant protein are present in equal amounts, but the SAO protein does not transport anions or bind stilbenedisulfonates with high affinity. We find that the rate constant for 35SO4(2-) efflux from SAO cells is about 50% that of normal cells, but the time course is a single exponential, indicating that there is no detectable heterogeneity in the distribution of SAO band 3 in the population of cells. Treatment of intact cells with the homobifunctional crosslinker BS3 (bis[sulfosuccinimido]suberate) produces similar amounts of covalent dimer in both normal and SAO cells. In SAO cells, copies of normal band 3 can be distinguished from SAO band 3 by treating with H2DIDS to form a crosslink between major chymotryptic fragments (60 kDa and 35 kDa) within one subunit. Successive treatment of cells with [3H]-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate ([3H]H2DIDS), BS3, and chymotrypsin gives 3H-labeled products that include homodimer of normal band 3 as well as products of crosslinking normal band 3 with the 60- and 35-kDa fragment of SAO band 3. The results are in semiquantitative agreement with a model in which covalent dimer forms between normal and SAO subunits with the same probability as between two normal subunits. These results indicate that the normal copy of band 3, complexed in a heterodimer with SAO band 3, reacts with H2DIDS as in normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Artificial metalloenzymes are generated by forming a novel active site on the dimer interface of the transcription factor LmrR. Two copper centers are incorporated by binding to ligands in each half of the dimer. With this system up to 97 % ee was obtained in the benchmark CuII catalyzed Diels–Alder reaction.

Photosynthetic reaction centres of green sulphur bacteria and of heliobacteria show a remarkable similarity to photosystem 1 of O2-evolving photosynthesis. Three features are common to this ‘reaction centre 1-type’. (1) A redox potential negative enough to reduce ferredoxin is generated. (2) Iron-sulphur centres are constituents of the bound electron acceptor complex. (3) A dimer of large, very hydrophobic protein subunits not only binds the redox centres that are involved in the initial steps of charge separation, but also binds the pigments of the inner light antenna. This protein dimer is a heterodimer in photosystem I, but appears to be a homodimer in reaction centres of green sulphur bacteria and of heliobacteria. The dimer-forming proteins contain a highly conserved dodecapeptide to which one of the iron-sulphur centres is bound.

The ‘softness’ of the electrospray ionization (ESI) method provides a direct link between solution chemistry and the inherent gas-phase environment of mass Spectrometry. Available results related to the preservation of non-covalent associations into the gas phase after ESI are reviewed. These associations include the possible retention of elements of higher order protein structure, non-covalent polypeptide–heme associations and enzyme complexes. Experimental results are presented showing that non-covalently bound polypeptide and protein dimer ions are relatively common as low level contributions to ESI mass spectra. It is argued that these dimers are reflective of multimeric species in solution since Coulombic barriers preclude dimerization after ESI although uncertainty remains regarding whether they exist prior to the formation of highly charged droplets. The dissociation of dimers is facile and for proteins can yield monomers having a broad distribution of charge states. The detection of non-covalently associated dimers requires gentle ESI mass spectrometer interface conditions, yielding relatively low levels of internal excitation. Under such conditions incomplete molecular ion desolvation can result in experimental artifacts for tandem mass spectrometric experiments. ESI mass Spectrometry may have broad potential for the study of noncovalent liquid phase associations.