Zero-length Cross-linker Research Articles

Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding

The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c 6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1–79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His 6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I Ref. [5] and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50–65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and 15N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.

Conjugation reactions in the preparations of quantum dot-based immunoluminescent probes for analysis of proteins by capillary electrophoresis

A number of biologically important molecules, such as DNA, proteins, and antibodies, are routinely conjugated with fluorescent tags for high-sensitivity analyses. Here, the application of quantum dots in the place of bright and size-tunable luminophores is studied. Several selected bioconjugation reactions via zero-length cross-linkers, long-chain linkers, and oriented methods for linking of quantum dots with proteins were tested. Anti-ovalbumin, anti-proliferating cell nuclear antigen, anti-hemagglutinin, and anti-CD3 membrane protein as model antibodies and annexin V were used as high-specificity selectors. The reaction yield and efficiency of the prepared immunoluminescent probes were tested by capillary zone electrophoresis with laser-induced fluorescence detection.

Nanotubes with hybrid CdTe nanocrystals: Fabrication, property, and surface functional decoration for bioapplications

We have demonstrated a two-step synthesis for preparing morphology- and luminescence-tunable fibers that uses green-emitting CdTe nanocrystals (NCs), Cd2+, thioglycolic acid (TGA), and tetraethyl orthosilicate (TEOS). Hybrid CdTe NCs consisted of a CdTe core and CdS-like clusters in the fibers were created during the synthesis. The CdTe NCs were first coated with a very thin SiO2 shell containing Cd2+ and TGA molecules in an alkaline condition. The SiO2-coated CdTe NCs were assembled together with Cd–TGA clusters for the formation of the fibers. A subsequent reflux process enabled the creation of fibers and caused the formation of CdS-like clusters in the vicinity of CdTe NCs in the fibers. Because of these clusters closed to the CdTe NCs, their effective size increases to reduce the quantum size effect. This special core-shell structure was found to have extraordinary properties: a narrowed photoluminescence (PL) spectrum, a red-shifted PL peak, and a high PL efficiency. The morphology of the fibers (tubal and solid) depends on the concentrations of TGA, Cd2+ ions, and TEOS in the precursor solution together with the concentration of CdTe NCs in a subsequent reflux. These fibers, especially for nanotubes, exhibited not only a tunable morphology and emitting color but also a functional surface, making them well suited for a variety of applications, such as bio-probing and drug delivery. The nanotubes were conjugated with immunoglobulin G (IgG) by using a zero-length cross linker because of carboxyl groups on their surface. The SiO2 component in the nanotubes enables bio-conjugation with IgG through other functional groups, making these nanotubes amenable for bioapplications.

Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira

Leptospirosis constitutes a major health problem in tropical and subtropical countries and is caused by pathogenic Leptospira. Immuno-magnetic separation (IMS) is considered to be an effective pre-enrichment method to isolate Leptospira from liquid specimen. We applied an inexpensive and simple IMS protocol using zero-length cross-linkers to immobilize polyclonal anti-leptospiral antibodies onto magnetic particles. The IMS-system has been optimized and evaluated by the assessment of the capture efficiency (CE). Main parameters that influence the conjugation procedure were optimized, including the amount of protein per milligram of magnetic particles, the pH and ionic strength of the conjugation buffer. The bead-bound leptospiral fraction was identified by using acridine orange fluorescence dye. The highest value for CE occurred when using high molar phosphate saline buffer at a pH around the isoelectric point of the antibodies. Finally, up to 3×108 leptospiral cells per mL could have been captured with approximately 50 μg of antibody-labelled particles. Strong particle agglutination could be observed during incubation for leptospiral concentrations in the range of 107–108 cells per mL. Despite covalent binding, we show that the physical adsorption parameters pH and ionic strength of the conjugation buffer greatly affect the entire immobilization process with regard to the CE, thus being able to increase the reactivity of the particles. We therefore conclude that a well-adjusted conjugation buffer for the used chemistry could possibly replace expensive and more complicated antibody immobilization methods.

A novel cell support membrane for skin tissue engineering: Gelatin film cross-linked with 2-chloro-1-methylpyridinium iodide

A novel method for preparing cell support membrane which employs heterogeneously cross-linking (CR) process to prepare water-insoluble gelatin (GEL) films was suggested, and compared with 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide. In this study, we found that 2-chloro-1-methylpyridinium iodide (CMPI) is an effective zero-length cross-linker of gelatin, and showed a superior activation ability of carboxylic acid sodium salt under heterogeneous reaction. The CMPI-CR-GEL films showed high water uptake ability, reasonable biodegradability, and excellent cytocompatibility. In vitro studies with 3T3 fibroblasts demonstrated an increased cell viability for those cells grown on CMPI-CR-GEL membranes, and significantly increased cell numbers from Day 1–12. Furthermore, under heterogeneous process, the gelatin film can fabricate in advance, and maintained the original dimension, thickness, and shape after cross-link. Meanwhile, the reaction residues can be easy removed by rinsing and washing processes. These characteristics make the process easily transfer to industrial manufacturing in the future.

Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein

Intracellular Ca2+-dependent cellular responses are often mediated by the ubiquitous protein CaM (calmodulin), which, upon binding Ca2+, can interact with and alter the function of numerous proteins. In the present study, using a newly developed functional proteomic screen of rat brain extracts, we identified PRG-1 (plasticity-related gene-1) as a novel CaM target. A CaM-overlay and an immunoprecipitation assay revealed that PRG-1 is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance and zero-length cross-linking showed that the C-terminal putative cytoplasmic domain (residues 466-766) of PRG-1 binds equimolar amounts of CaM in a Ca2+-dependent manner, with a relatively high affinity (a Kd value for Ca2+/CaM of 8 nM). Various PRG-1 mutants indicated that the Ca2+/CaM-binding region of PRG-1 is located between residues Ser554 and Gln588, and that Trp559 and Ile578 potentially anchor PRG-1 to CaM. This is supported by pronounced changes in the fluorescence emission spectrum of Trp559 in the PRG-1 peptide (residues 554-588) upon binding to Ca2+/CaM, showing the stoichiometrical binding of the PRG-1 peptide with Ca2+/CaM. Immunoblot analyses revealed that the PRG-1 protein is abundant in brain, but is weakly expressed in the testes. Immunohistochemical analysis revealed that PRG-1 is highly expressed in forebrain structures and in the cerebellar cortex. Furthermore, PRG-1 localizes at the postsynaptic compartment of excitatory synapses and dendritic shafts of hippocampal neurons, but is not present in presynaptic nerve terminals. The combined observations suggest that PRG-1 may be involved in postsynaptic functions regulated by intracellular Ca2+-signalling.

Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons

CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1–179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178–229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

Synthesis and characterization of diethylenetriaminepentaacetic acid-chitosan-coated cobalt ferrite core/shell nanostructures

Special diethylenetriaminepentaacetic acid (DTPA)-chitosan-coated cobalt ferrite core/shell nanoparticles have been synthesized via a novel zero-length emulsion crosslinking process and characterized via crosslinking degree, simultaneous thermogravimetric analysis and differential scanning calorimetry, X-ray diffractometry, Fourier transform infrared spectrometer, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and vibration sample magnetometry. The experimental results showed that the CoFe 2O 4 nanoparticles were really encapsulated with a DTPA-chitosan hybrid layer and the nanocomposites were proved to be nearly superparamagnetic with saturation magnetization of 26.6 emu g −1.

Mapping of Drebrin Binding Site on F-Actin

Drebrin is a filament-binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full-length protein. Site-directed mutagenesis, electron microscopic reconstruction, and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and MTS1 [1,1-methanediyl bis(methanethiosulfonate)]. Site-directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 A from C374 of actin protomer 1 and that native cysteine 308 of drebrin is near C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, can link the N-terminal G-S extension of the recombinant DrABD to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different lengths (5.4-19 A). These results suggest that the "core" DrABD is centered on actin subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of electron microscopic reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin-binding proteins.

Comparative Kinetics of the ATPase and Actin Sliding Velocity of Myosin Isoforms

Myosin isoform expression varies according to demand and pathology, and the kinetics of the resultant actomyosin motor protein determine maximal sarcomere shortening velocity. Studies of muscle fibers and isolated myosin isoforms have shown that actin sliding velocity correlates with ATP hydrolysis. We studied this relationship for isoforms of actomyosin complexes and examined ATP hydrolysis and the effect of association and dissociation of myosin with actin. Sliding velocity of actin filaments was measured in motility assays with different myosin isoforms. Actin-dependent ATP hydrolysis rate of isolated myosin sub-fragments interacting with filamentous actin, or cross-linked with actin by the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were measured. ATPase activity and velocity of actin moved by myosin isoforms from rat and canine cardiac and skeletal muscle were measured at 25°, 30° and 35°C. Motility velocity plotted against ATPase activity of different myosin isoforms showed a linear correlation with a slope of 330 ±20 (nm/sec)/(ATP/sec) ( R2 = 0.98); the slope coefficient was 19% of the slope of the relationship for intact muscle described by M. Barany (J Gen Physiol, 1967) across a wide range of temperatures. Activation energy of sliding velocity (92 - 96 kJ/mol) and ATPase rate (83 - 121 kJ/mol) of different myosin isoforms were similar. Cross-linking of actomyosin complexes by EDC increased ATP hydrolysis rate 4-fold above ATPase at saturating [Actin]. This suggests that association/dissociation kinetics are rate-limiting and that ATPase is activated in maximally 25% myosin molecules interacting with actin in solution. The calculated displacement of actin filaments (D = 330 nm) per ATP hydrolyzed under the experimental conditions used here suggests that unloaded cross-bridges may displace actin over a multitude of the minimal steps of 2.7 nm that can be made by myosin along the actin filaments.

Preparation of chitosan–ethylenediaminetetraacetate-enwrapped magnetic CoFe 2O 4 nanoparticles via zero-length emulsion crosslinking method

A kind of magnetic multiple functional groups nanocomposites, chitosan–ethylenediaminetetraacetate (EDTA)-enwrapped CoFe 2O 4 nanoparticles, i.e. CoFe 2O 4@chitosan–EDTA nanocomposites were synthesized by a facile zero-length emulsion crosslinking process. In this method, CoFe 2O 4 was used as magnetic core, and 1-ethyl-3-(3-dimethylminopropyl) carbodiimide hydrochloride (EDAC) was used as a crosslinker, integrating amino group of chitosan and carboxyl group of EDTA. Determination of amino groups in chitosan modified by EDAC-activated EDTA was carried out through the trinitrobenzenesulfonic acid (TNBS) method. The as-prepared magnetic nanocomposites were characterized by XRD, FT-IR, XPS, SEM, EDS, TEM, SAED and vibrating sample magnetometer (VSM), and the results showed that the as-prepared CoFe 2O 4@chitosan–EDTA nanocomposites have good dispersibility, spherical shape and enough magnetization. The method proposed can be extended to fabricate other magnetic nanocomposites possessed amino and carboxyl groups.

Selective adsorption of surface-modified ferritin on a phase-separated polymer blend

We compare the adsorption behavior of wild and surface-modified ferritin on a homopolymer blend of poly(desaminotyrosyl tyrosine dodecyl ester carbonate) (PDTD) and poly(ɛ-caprolactone) (PCL). Wild ferritin was alkylated by zero length cross-linking to surface carboxylate groups activated by water soluble carbodiimide (EDC). Modification was confirmed by anion exchange chromatography. All the adsorption experiments were done using deionized water (pH 5.8) and ultramicrotomed thin polymer films of thickness ∼100 nm. Transmission electron microscopy shows a clear selectivity of alkylated ferritin adsorption onto the PCL phase while wild ferritin predominantly adsorbs onto the PDTD phase. We attribute these differences to the nature of the electrostatic interaction between the two types of ferritin and the polymer surface.

Identification and Characterization of Wolframin, the Product of the Wolfram Syndrome Gene (WFS1), as a Novel Calmodulin-Binding Protein

To search for calmodulin (CaM) targets, we performed affinity chromatography purification of a rat brain extract using CaM fused with GST as the affinity ligand. Proteomic analysis was then carried out to identify CaM-binding proteins. In addition to identifying 36 known CaM-binding proteins, including CaM kinases, calcineurin, nNOS, the IP(3) receptor, and Ca(2+)-ATPase, we identified an ER transmembrane protein, wolframin [the product of the Wolfram syndrome gene (WFS1)] as interacting. A CaM overlay and an immunoprecipitation assay revealed that wolframin is capable of binding the Ca(2+)/CaM complex in vitro and in transfected cells. Surface plasmon resonance analysis and zero-length cross-linking showed that the N-terminal cytoplasmic domain (residues 2-285) of wolframin binds to an equimolar unit of CaM in a Ca(2+)-dependent manner with a K(D) for CaM of 0.15 muM. Various truncation and deletion mutants showed that the Ca(2+)/CaM binding region in wolframin is located from Glu90 to Trp186. Furthermore, we demonstrated that three mutations (Ala127Thr, Ala134Thr, and Arg178Pro) associated with Wolfram syndrome completely abolished CaM binding of wolframin. This observation may indicate that CaM binding is important for wolframin function and that impairment of this interaction by mutation contributes to the pathology seen in Wolfram syndrome.

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization(1). Mechanistically, PICUP involves photo-oxidation of Ru(2+) in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru(3+) by irradiation with visible light in the presence of an electron acceptor. Ru(3+) is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical(1,2). Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods(3,4) include short (<or=1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid beta-protein variants (Abeta40 and Abeta42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (≤1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid β-protein variants (Aβ40 and Aβ42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Functional Halt Positions of Rotary F OF 1-ATPase Correlated with Crystal Structures

The F OF 1-ATPase is a rotary molecular motor. Driven by ATP-hydrolysis, its central shaft rotates in 80° and 40° steps, interrupted by catalytic and ATP-waiting dwells. We recorded rotations and halts by means of microvideography in laboratory coordinates. A correlation with molecular coordinates was established by using an engineered pair of cysteines that, under oxidizing conditions, formed zero-length cross-links between the rotor and the stator in an orientation as found in crystals. The fixed orientation coincided with that of the catalytic dwell, whereas the ATP waiting dwell was displaced from it by +40°. In crystals, the convex side of the cranked central shaft faces an empty nucleotide binding site, as if holding it open for arriving ATP. Functional studies suggest that three sites are occupied during a catalytic dwell. Our data imply that the convex side faces a nucleotide-occupied rather than an empty site. The enzyme conformation in crystals seems to differ from the conformation during either dwell of the active enzyme. A revision of current schemes of the mechanism is proposed.

Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.

Structural studies of hen egg-white lysozyme dimer: Comparison with monomer

A facile method for the formation of covalent bonds between protein molecules is zero length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, we report a cross-linking method for lysozyme and some structural studies as well as catalytic activity assay was performed on lysozyme dimer. The results showed that catalytic activity of lysozyme dimer was the same as monomer. Also, the GdnCl-induced equilibrium unfolding of hen egg-white lysozyme monomer and dimer at pH 2 was studied over a temperature range of 290.7–303.2 K by means of CD spectroscopy. The lack of coincidence between two unfolding curves at 222 and 289 nm in lysozyme dimer was observed, which suggested the existence of intermediate state in unfolding process, while lysozyme monomer showed a single cooperative transition. Thus, the thermodynamic parameters were estimated on the basis of two-state mechanism for lysozyme monomer and three-state one for lysozyme dimer. These results indicated that zero length cross-linking can stabilize the intermediate, so the population of intermediate increased. Our results offer a special opportunity to study the role of intermediates in protein folding mechanisms. In addition thermal unfolding of monomer and dimer in 222 nm was achieved.

Fine structure of the promoter–σ region 1.2 interaction

We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the -10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the sigma subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with sigma1.2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases at the junction of the -10 element and the discriminator region in an unregulated rRNA promoter variant and in the lambdaP(R) promoter. Our studies indicate that nontemplate strand bases adjacent to the -10 element bind within a 9-aa interval in sigma1.2 (residues 99-107). We also demonstrate that the downstream-most base on the nontemplate strand of the -10 hexamer cross-links to sigma region 2, and not to sigma1.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation.

Structural Evidence for Co-Evolution of the Regulation of Contraction and Energy Production in Skeletal Muscle

Skeletal muscle phosphorylase kinase (PhK) is a Ca2+-dependent enzyme complex, (αβγδ)4, with the δ subunit being tightly bound endogenous calmodulin (CaM). The Ca2+-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the “excitation–contraction–energy production triad.” Although the Ca2+-dependent protein–protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic γ and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the γ subunit sharing high sequence similarity with the Ca2+-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca2+ stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca2+ of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca2+-binding proteins troponin C and CaM.