A mercuric ion uptake role for the integral inner membrane protein, MerC, involved in bacterial mercuric ion resistance.
AuthorsSahlman L, Wong W, Powlowski J
JournalJ Biol Chem
PubMed ID9368013
Bacterial detoxification of mercuric ion depends on the presence of one or more integral membrane proteins (MerT and/or MerC) whose postulated function is in transport of Hg2+ from a periplasmic Hg2+-binding protein (MerP) to cytoplasmic mercuric reductase. In this study, MerC from the Tn21-encoded mer operon was overexpressed and studied ... More
Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C.
AuthorsLeszyk J, Collins JH, Leavis PC, Tao T
JournalBiochemistry
PubMed ID3427058
We have used the sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) to study the interaction of rabbit skeletal muscle troponin C (TnC) and troponin I (TnI). TnC was specifically labeled at Cys-98 by the maleimide moiety of BPMal, and a binary complex was formed with TnI in the presence of Ca2+. ... More
Mapping sonic hedgehog-receptor interactions by steric interference.
AuthorsPepinsky RB, Rayhorn P, Day ES, Dergay A, Williams KP, Galdes A, Taylor FR, Boriack-Sjodin PA, Garber EA
JournalJ Biol Chem
PubMed ID10753901
'We have defined regions in the Sonic hedgehog (Shh) molecule that are important for Patched (Ptc) receptor binding by targeting selected surface amino acid residues with probes of diverse sizes and shapes and assessing the effects of these modifications on function. Eleven amino acid residues that surround the surface of ... More
Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin.
AuthorsGraceffa P
JournalJ Biol Chem
PubMed ID8530428
'The NH2- and COOH-terminal domains of muscle caldesmon are separated by a long alpha-helical stretch. Cys-580, in the COOH-terminal domain, can be rapidly and efficiently disulfide-cross-linked to Cys-374 of actin by incubation with actin modified with 5,5''-dithiobis(2-nitrobenzoic acid) (Graceffa, P., and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310). Upon ... More
Smooth muscle alpha-tropomyosin crosslinks to caldesmon, to actin and to myosin subfragment 1 on the muscle thin filament.
AuthorsGolitsina NL, Lehrer SS
JournalFEBS Lett
PubMed ID10601656
'To obtain proximity information between tropomyosin (Tm) and caldesmon (CaD) on the muscle thin filament, we cloned gizzard alphaTm and created two single Cys mutants S56C/C190S (56Tm) and D100C/C190S (100Tm). They were labeled with benzophenone maleimide (BPM) and UV-irradiated on thin filaments. One chain of BPM-56Tm and two chains of ... More
A recombinant monocysteine mutant (Ser to Cys-155) of fast skeletal troponin T: identification by cross-linking of a domain involved in a physiologically relevant interaction with troponins C and I.
AuthorsJha PK, Sarkar S
JournalBiochemistry
PubMed ID9724539
'Troponin T (TnT), a subunit of the heterotrimeric troponin (Tn) complex, is essential for the Ca2+ regulation of vertebrate striated muscle contraction both in vivo and in vitro. With the exception of bovine cardiac TnT, all known vertebrate TnT isoforms lack a thiol group, a property which makes the wild-type ... More
Relaxation time, interthiol distance, and mechanism of action of ribosomal protein S1.
AuthorsOdom OW, Deng HY, Subramanian AR, Hardesty B
JournalArch Biochem Biophys
PubMed ID6201138
'The two sulfhydryl groups of ribosomal protein S1 from Escherichia coli have been labeled with fluorescent maleimides and the distance between them has been determined by nonradiative energy transfer. This distance was found to be approximately 27 A for both free S1 and S1 bound to 30 S subunits. This ... More
Design of a photoreactive analogue of the Escherichia coli heat-stable enterotoxin STIb: use in identifying its receptor on rat brush border membranes.
AuthorsGariépy J, Schoolnik GK
JournalProc Natl Acad Sci U S A
PubMed ID3510436
'The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography. This analogue was iodinated and shown to bind specifically to rat intestinal membranes. The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate. ... More
Topological analysis of the peripheral benzodiazepine receptor in yeast mitochondrial membranes supports a five-transmembrane structure.
AuthorsJoseph-Liauzun E, Delmas P, Shire D, Ferrara P
JournalJ Biol Chem
PubMed ID9442055
'The peripheral benzodiazepine receptor, implicated in the transport of cholesterol from the outer to the inner mitochondrial membrane, is predicted by hydropathy analysis to feature five membrane-spanning domains, with the amino terminus within the mitochondrial periplasm and the carboxyl terminus in the external cytoplasm. We have tested these structural predictions ... More
Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.
AuthorsTao T, Gong BJ, Leavis PC
JournalScience
PubMed ID2138356
'The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between ... More
Activating amphiphiles cause a conformational change of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes according to proteolytic digestion.
AuthorsLi L, Karlsson OP, Wieslander A
JournalJ Biol Chem
PubMed ID9368025
'1,2-Diacylglycerol 3-glucosyltransferase synthesizes the major nonbilayer-prone lipid monoglucosyldiacylglycerol (MGlcDAG) in the membrane of Acholeplasma laidlawii, which is important for the spontaneous curvature, and is a regulatory site for the lipid surface charge density. A potential connection between activity and a conformational change of this enzyme, governed by essential lipid activators, ... More
Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex.
AuthorsXu T, Vasilyeva E, Forgac M
JournalJ Biol Chem
PubMed ID10506135
'The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from ... More
Identification of the photocrosslinking sites in troponin-I with 4-maleimidobenzophenone labelled mutant troponin-Cs having single cysteines at positions 158 and 21.
AuthorsLeszyk J, Tao T, Nuwaysir LM, Gergely J
JournalJ Muscle Res Cell Motil
PubMed ID9682135
'Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypeptide chains, in agreement with other studies using fragments ... More
Site-specific protein modification to identify the MutL interface of MutH.
AuthorsToedt GH, Krishnan R, Friedhoff P
JournalNucleic Acids Res
PubMed ID12560476
'We have mapped the region for the protein interaction site of the Escherichia coli mismatch repair protein MutH for its activator protein MutL by a site-specific protein modification approach. For this purpose we generated a cysteine-free variant of MutH and 12 variants thereof, each containing a single cysteine residue at ... More
Calmodulin interaction with the skeletal muscle sarcoplasmic reticulum calcium channel protein.
AuthorsYang HC, Reedy MM, Burke CL, Strasburg GM
JournalBiochemistry
PubMed ID8286381
'Studies were initiated to define the equilibria of calmodulin binding to the skeletal muscle sarcoplasmic reticulum (SR) Ca(2+)-release channel protein in native SR vesicles. Calmodulin affinity-labeling experiments indicated that the major calmodulin receptor in heavy SR preparations was a protein of M(r) > 450,000, corresponding to the Ca(2+)-release channel protein. ... More
The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions.
AuthorsMcLachlin DT, Bestard JA, Dunn SD
JournalJ Biol Chem
PubMed ID9614129
'An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity ... More
The conformation of the C-terminal region of actin: a site-specific photocrosslinking study using benzophenone-4-maleimide.
AuthorsTao T, Lamkin M, Scheiner CJ
JournalArch Biochem Biophys
PubMed ID4026298
'The site-specific photocrosslinker, benzophenone-4-maleimide, was used to label G-actin specifically at Cys-374, the penultimate residue from the C terminus. The resultant BP-G-actin was polymerized to form BP-F-actin, and both forms of actin were irradiated to activate the benzophenone moiety. We found that for BP-F-actin both intersubunit and intrasubunit photocrosslinks were ... More
Formation and properties of smooth muscle myosin 20-kDa light chain-skeletal muscle myosin hybrids and photocrosslinking from the maleimidylbenzophenone-labeled light chain to the heavy chain.
AuthorsRajasekharan KN, Morita JI, Mayadevi M, Ikebe M, Burke M
JournalArch Biochem Biophys
PubMed ID1832844
'Experimental conditions which permit the exchange of smooth muscle 20-kDa light chain into skeletal muscle myosin are described. The hybridization does not result in the regulation of actin-activated ATPase activity of the hybrid myosin by smooth light chain phosphorylation. Further, the KCl dependence of the Mg-ATPase activity of the hybrid ... More
Identification of the site of photocross-linking formed in the absence of magnesium nucleotide from SH2 (Cys-697) in myosin subfragment 1 labeled with 4'-maleimidylbenzophenone.
AuthorsAgarwal R, Rajasekharan KN, Burke M
JournalJ Biol Chem
PubMed ID1989981
'The site of photocross-linking between Cys-697 (SH2), prelabeled with 4''-[14C]maleimidylbenzophenone, and the 50-kDa segment of myosin S1 on irradiation in the absence of nucleotide has been determined by isolation of the 20-50-kDa adduct and subsequent tryptic proteolysis. Isolation and partial sequencing of the radioactively labeled peptide indicate that the site ... More
The amino-terminal fragment of gelsolin is cross-linked to Cys-374 of actin in the EGTA-resistant actin-gelsolin complex.
AuthorsDoi Y, Kanatani Y, Kim F
JournalFEBS Lett
PubMed ID1333414
It has been shown that the EGTA-resistant actin, one of the two actin molecules associated to gelsolin, can be predominantly cross-linked to gelsolin by benzophenone-4-maleimide (BPM), a photoaffinity-labeling reagent, which was conjugated to Cys-374 of actin prior to cross-linking (Doi, Y., Banba, M. and Vertut-Doï, A. (1991) Biochemistry 30, 5769-5777). ... More
Benzophenone photophores in biochemistry.
AuthorsDormán G, Prestwich GD
JournalBiochemistry
PubMed ID8180191
The photoactivatable aryl ketone derivatives have been rediscovered as biochemical probes in the last 5 years. The expanding use of benzophenone (BP) photoprobes can be attributed to three distinct chemical and biochemical advantages. First, BPs are chemically more stable than diazo esters, aryl azides, and diazirines. Second, BPs can be ... More
Photocrosslinking of calmodulin and/or actin to chicken gizzard caldesmon.
AuthorsWang CL
JournalBiochem Biophys Res Commun
PubMed ID3190671
The two sulfhydryl groups of chicken gizzard caldesmon were specifically labeled with a photoreactive crosslinker, benzophenone-maleimide, to study its interactions with calmodulin and/or actin. When incubated with F-actin caldesmon crosslinks to a single actin monomer; it can, however, crosslink to up to two calmodulin molecules in the presence, but not ... More
Site-specific photo-cross-linking studies on interactions between troponin and tropomyosin and between subunits of troponin.
AuthorsTao T, Scheiner CJ, Lamkin M
JournalBiochemistry
PubMed ID3099834
We have used the sulfhydryl-specific heterobifunctional photo-cross-linker 4-maleimidobenzophenone (BP-Mal) to study the interactions of rabbit skeletal tropomyosin with troponin and of the troponin subunits with each other. We found that alpha,alpha-tropomyosin specifically labeled at Cys-190 with BP-Mal photo-cross-links with all three subunits of troponin with decreasing cross-linking yields in the ... More
Evidence for contact between the cyclic AMP receptor protein and the delta 70 subunit of Escherichia coli RNA polymerase.
AuthorsJin R, Sharif KA, Krakow JS
JournalJ Biol Chem
PubMed ID7642591
The loop at the 52-position of the cAMP receptor protein (CRP) has been suggested as a potential site for contacting RNA polymerase on Class II promoters where the CRP binding site is located at position -41.5 (Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., ... More
Photoimmobilization of a bioactive laminin fragment and pattern-guided selective neuronal cell attachment.
AuthorsClémence JF, Ranieri JP, Aebischer P, Sigrist H
JournalBioconjug Chem
PubMed ID7578361
To attain light-dependent functionalization of biocompatible materials, a photolabel-derivatized, bioactive laminin fragment has been synthesized, chemically characterized, and photoimmobilized. Covalent high-resolution patterning of the laminin fragment CDPGYIGSR to hydroxylated fluorinated ethylene propylene (FEP-OH), poly(vinyl alcohol), and glycophase glass has been achieved. The synthetic peptide CDPGYIGSR was thermochemically coupled to either ... More
Photopatterning of antibodies on biosensors.
AuthorsLiu XH, Wang HK, Herron JN, Prestwich GD
JournalBioconjug Chem
PubMed ID11087322
The immobilization of biomolecules on surfaces in defined micropatterns has become increasingly important for the development of new diagnostic devices and high-throughput genetic and drug screening protocols. We describe the synthesis and testing of thiol-reactive, photoactivatable linkers that will permit laser micropatterning or photolithographic patterning of surfaces. In these linkers, ... More
Covalent binding of peptides to the N-terminal hydrophobic region of cardiac troponin C has limited effects on function.
AuthorsLin X, Dotson DG, Putkey JA
JournalJ Biol Chem
PubMed ID8550567
Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide ... More
Localization of Cys133 of rabbit skeletal troponin-I with respect to troponin-C by resonance energy transfer.
AuthorsLuo Y, Wu JL, Gergely J, Tao T
JournalBiophys J
PubMed ID9635764
We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our ... More
Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T.
AuthorsLeszyk J, Collins JH, Leavis PC, Tao T
JournalBiochemistry
PubMed ID3196696
The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. ... More
Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90.
AuthorsBasu S, Basu A, Modak MJ
JournalBiochem J
PubMed ID7506526
Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone ... More
Photo-cross-linking of rabbit skeletal troponin I deletion mutants with troponin C and its thiol mutants: the inhibitory region enhances binding of troponin I fragments to troponin C.
AuthorsJha PK, Mao C, Sarkar S
JournalBiochemistry
PubMed ID8780504
Contraction of vertebrate striated muscle is regulated by the strong Ca(2+)-dependent interaction between troponin I (TnI) and troponin C (TnC). To critically evaluate this interaction, we generated four recombinant deletion fragments of rabbit fast skeletal TnI: the NH2-terminal fragment (TnI1-94), the NH2 terminus and the inhibitory region (TnI1-120), the inhibitory ... More
Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4-(succinimidyl)benzophenone].
AuthorsWang J, Bauman S, Colman RF
JournalJ Biol Chem
PubMed ID10681528
Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the ... More
Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins.
AuthorsLuo Y, Wu JL, Li B, Langsetmo K, Gergely J, Tao T
JournalJ Mol Biol
PubMed ID10677290
The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant ... More
ATP synthase complex. Proximities of subunits in bovine submitochondrial particles.
AuthorsBelogrudov GI, Tomich JM, Hatefi Y
JournalJ Biol Chem
PubMed ID7836433
The catalytic sector, F1, and the membrane sector, F0, of the mitochondrial ATP synthase complex are joined together by a 45-A-long stalk. Knowledge of the composition and structure of the stalk is crucial to investigating the mechanism of conformational energy transfer between F0 and F1. This paper reports on the ... More
The stalk region of the Escherichia coli ATP synthase. Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer.
AuthorsWatts SD, Tang C, Capaldi RA
JournalJ Biol Chem
PubMed ID8910457
The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification ... More
Dynamic structure of the calmodulin-binding domain of the plasma membrane Ca-ATPase in native erythrocyte ghost membranes.
AuthorsYao Y, Gao J, Squier TC
JournalBiochemistry
PubMed ID8810906
We have used frequency-domain fluorescence resonance energy transfer (FRET) and anisotropy measurements to identify the structural properties of wheat germ calmodulin (CaM) bound to either the plasma membrane Ca-ATPase (PM-Ca-ATPase) in native erythrocyte ghost membranes or a peptide (C25W) that has an identical sequence to the CaM-binding domain on the ... More
Photoaffinity labeling of rat liver glutathione S-transferase, 4-4, by glutathionyl S-[4-(succinimidyl)-benzophenone].
AuthorsWang J, Bauman S, Colman RF
JournalBiochemistry
PubMed ID9843371
Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and xenobiotic substrate, was synthesized and shown to act as a photoaffinity label of rat liver glutathione S-transferase, 4-4. A time-dependent photoinactivation occurs upon irradiation at long wavelength UV light of the complex of enzyme and GS-Succ-BP. The rate of ... More
Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I. Photocross-linking and fluorescence studies using a recombinant troponin-C.
AuthorsWang ZY, Sarkar S, Gergely J, Tao T
JournalJ Biol Chem
PubMed ID2180953
We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine. TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with ... More
Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase.
AuthorsMcLachlin DT, Coveny AM, Clark SM, Dunn SD
JournalJ Biol Chem
PubMed ID10747904
The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, ... More
Adenosine 5'-0-[S-(4-succinimidyl-benzophenone)thiophosphate]: a new photoaffinity label of the allosteric ADP site of bovine liver glutamate dehydrogenase.
AuthorsMadhusoodanan KS, Colman RF
JournalBiochemistry
PubMed ID11327816
By reaction of adenosine 5'-monothiophosphate with benzophenone-4-maleimide, we synthesized adenosine 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (AMPS-Succ-BP) as a photoreactive ADP analogue. Bovine liver glutamate dehydrogenase is known to be allosterically activated by ADP, but the ADP site has not been located in the crystal structure of the hexameric enzyme [Peterson, P. E., and Smith, ... More
Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum.
AuthorsStrasburg GM, Hogan M, Birmachu W, Thomas DD, Louis CF
JournalJ Biol Chem
PubMed ID2961748
Wheat germ calmodulin (CaM) was derivatized at its single cysteine (Cys27) with either the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-EDANS) or the photoactivable cross-linker benzophenone-4-maleimide. Comparison of the native and derivatized wheat germ CaMs with native bovine testis CaM indicates that the concentrations of these proteins required for half-maximal stimulation of ... More
Proximity of regulatory light chains in scallop myosin.
AuthorsHardwicke PM, Szent-Györgyi AG
JournalJ Mol Biol
PubMed ID3159905
The distance between the regulatory light chains of the two heads of the scallop myosin molecule was estimated with the aid of two photolabile cross-linkers, benzophenone maleimide and p-azidophenacylbromide. These cross-linkers selectively alkylate thiol groups and have a maximum length of about 9 A. One of the two regulatory light ... More