Abstract:
:A new bifunctional spin-label (BSL) has been synthesized that can be immobilized on the surface of proteins, allowing measurement of rotational motion of proteins by saturation-transfer electron paramagnetic resonance (STEPR). The spin-label contains a photoactivatable azido moiety, a cleavable disulfide, and a nitroxide spin with restricted mobility relative to the rest of the label. The label reacts with surface lysine residues modified with beta-mercaptopropionate. Bifunctional attachment is achieved by photoactivation of the azido group. Any spin-label that remains monofunctionally attached after photolysis is removed by reduction of the disulfide. Only bifunctionally attached BSL remains on the protein. Hemoglobin was used to test the utility of the BSL in STEPR by comparison with hemoglobin modified with maleimide spin-label (MSL), a commonly used standard for the STEPR technique. MSL is a monofunctional spin-label which is fortuitously immobilized by local protein structure within hemoglobin. The BSL labeling of hemoglobin did not significantly affect the quaternary structure of hemoglobin as determined by gel filtration chromatography. The conventional EPR spectra of the mono- and bifunctionally attached BSL-hemoglobin were similar to the MSL-hemoglobin spectrum, indicating that both forms of BSL were rigidly bound to hemoglobin. In contrast, the spectrum obtained by reaction of modified hemoglobin lysine residues with MSL indicated that these labels were highly mobile. The monofunctionally attached BSL was mobilized upon octyl glucoside addition whereas bifunctionally attached BSL was only slightly mobilized, suggesting that hydrophobic interactions immobilize the monofunctionally attached label on hemoglobin. The response of STEPR spectra of mono- and bifunctionally attached BSL-hemoglobin to changes in hemoglobin rotational correlation time was similar to the MSL-hemoglobin over the range of 10(-5)-10(-3) s. The spectra of bifunctionally attached BSL indicated slightly less motion than corresponding spectra for MSL or monofunctionally attached BSL. The new BSL is a good reporter of protein rotation and does not require unique protein structures for its immobilization on the protein. Thus, the BSL should be more generally applicable for STEPR studies of membrane protein rotation than existing monofunctional spin-labels.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Wilcox MD,Parce JW,Thomas MJ,Lyles DSdoi
10.1021/bi00476a014subject
Has Abstractpub_date
1990-06-19 00:00:00pages
5734-43issue
24eissn
0006-2960issn
1520-4995journal_volume
29pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide to hydrogen carbonate, and its role in maintaining pH balance has made it an attractive drug target. Steroidal sulfamate esters, inhibitors of the cancer drug target steroid sulfatase (STS), are sequestered in vivo by CA II in red blood cells...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi902178w
更新日期:2010-04-27 00:00:00
abstract::Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R.,...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi701581y
更新日期:2008-01-15 00:00:00
abstract::The physicochemical and catalytic properties of thioredoxin-T' are described. This complemented protein structure consists of a 1:1 complex between the inactive fragments thioredoxin-T-(1--73) and thioredoxin T-(74--108). These are generated by selective trypsin cleavage at Arg-73 in lysine-modified and denatured Esch...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00592a010
更新日期:1979-12-11 00:00:00
abstract::We have found that the atomic force microscope (AFM) can be used to image the "beads-on-a-string" chromatin structure in a normal air environment following adsorption onto a cover glass substrate. Individual nucleosome cores and linker DNA could be resolved clearly along chromatin fibers that were reconstituted using ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00084a002
更新日期:1993-08-24 00:00:00
abstract::Urea-induced unfolding of Na,K-ATPase from pig kidney and from shark salt gland was studied by electron paramagnetic resonance (EPR) spectroscopy of a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups which are essential for activity. Urea-induced structural changes lead to the inhibition of Na...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi901124j
更新日期:2009-09-29 00:00:00
abstract::Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor of the serpin superfamily which rapidly inactivates tissue plasminogen activator (tPA), but reacts with thrombin at a much slower rate. Based on the previous mutagenesis studies and the X-ray crystal structure of the thrombin E192Q-bovine pancrea...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9808518
更新日期:1998-09-22 00:00:00
abstract::The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Th...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi025967p
更新日期:2002-10-08 00:00:00
abstract::The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme. Although a global conformational change in ATCase upon the binding o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00467a019
更新日期:1990-04-17 00:00:00
abstract::In human glutathione transferase (GST) A1-1, the C-terminal region covers the active site and contributes to substrate binding. This region is flexible, but upon binding of an active-site ligand, it is stabilized as an amphipatic alpha-helix. The stabilization has implications for the catalytic activity of the enzyme....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi991482y
更新日期:1999-12-07 00:00:00
abstract::Protease inhibition by metastable serine protease inhibitors (serpins) is mediated by one of the largest functional intradomain conformational changes known in biology. In this extensive structural rearrangement, protease-serpin complex formation triggers cleavage of the serpin reactive center loop (RCL), its subseque...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.6b00839
更新日期:2017-01-31 00:00:00
abstract::The cAMP-dependent protein kinase (PKA) controls a large number of cellular functions. One critical PKA substrate in the brain and heart is the L-type Ca(2+) channel Ca(v)1.2, the activity of which is upregulated by PKA. The main PKA phosphorylation site is serine 1928 in the central pore forming alpha(1)1.2 subunit o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi051593z
更新日期:2006-03-14 00:00:00
abstract::The 1.9 A X-ray crystal structure of human myeloperoxidase complexed with cyanide (R = 0.175, R(free) = 0.215) indicates that cyanide binds to the heme iron with a bent Fe-C-N angle of approximately 157 degrees, and binding is accompanied by movement of the iron atom by 0.2 A into the porphyrin plane. The bent orienta...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0111808
更新日期:2001-11-20 00:00:00
abstract::Here, we introduce the concept of the "seleno effect" in the study of oxidoreductases that catalyze thiol/disulfide exchange reactions. In these reactions, selenium can replace sulfur as a nucleophile, electrophile, or leaving group, and the resulting change in rate (the seleno effect) is defined as kS/ kSe. In soluti...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.8b00004
更新日期:2018-03-20 00:00:00
abstract::Drug substrates stimulate ATPase activity of the P-glycoprotein (P-gp) ATP-binding cassette drug pump by an unknown mechanism. Cross-linking analysis was performed to test if drug substrates stimulate P-gp ATPase activity by altering cross-talk at the first transmission interface linking the drug-binding [intracellula...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.6b00233
更新日期:2016-05-24 00:00:00
abstract::The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstra...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi300693k
更新日期:2012-08-28 00:00:00
abstract::Eosinophil cationic protein (ECP) is a component of the eosinophil granule matrix. It shows marked toxicity against helminth parasites, bacteria single-stranded RNA viruses, and host epithelial cells. Secretion of human ECP is related to eosinophil-associated allergic, asthmatic, and inflammatory diseases. ECP belongs...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0264521
更新日期:2002-10-08 00:00:00
abstract::Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. sGC is activated by nitric oxide (NO) and therefore plays a central role in NO signal transduction. Activation of sGC by NO is believed to be mediated by the interaction between NO and the heme of sGC. Spectroscopic a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971825x
更新日期:1997-12-16 00:00:00
abstract::The pivotal role of the Arg-Gly-Asp (RGD) peptide motif in integrin-mediated cell adhesive interactions with extracellular matrix and plasma proteins stimulated the present design of nonpeptidic mimetics of this sequence. To probe the structural requirements for RGD recognition by integrins, we designed various struct...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00055a002
更新日期:1993-02-02 00:00:00
abstract::Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00587a019
更新日期:1979-10-02 00:00:00
abstract::The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melano...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi061549z
更新日期:2006-11-21 00:00:00
abstract::The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1α (hepatocyte nuclear factor-1α), while the PCD activity of DCoH1 homot...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi1015056
更新日期:2010-11-30 00:00:00
abstract::To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. A...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi026100u
更新日期:2002-11-19 00:00:00
abstract::4-Oxalocrotonate tautomerase (EC 5.3.2-; 4-OT), a hexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids, converting unconjugated ketones to the conjugated isomers via a dienolic intermediate. The recently solved crystal structure of an isozyme of 4-OT suggests that ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi951077g
更新日期:1996-01-23 00:00:00
abstract::A newly recognized type of protein-ligand interaction phenomenon has resulted in the preparation of simple, nonionic, and highly specific gel derivatives for selective adsorption chromatography. The essential structure of the immobilized ligand can be represented as agarose-CH2CH2SO2CH2CH2SCH2CH2OH, which was prepared...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00396a049
更新日期:1987-11-03 00:00:00
abstract::Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi002938a
更新日期:2001-08-07 00:00:00
abstract::Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi101650q
更新日期:2011-02-22 00:00:00
abstract::To study the function of exon 21 of the insulin receptor, a mutant human insulin receptor lacking this domain was constructed. The mutant HIR delta E21 cDNA was transfected into Rat-1 fibroblasts and stable cell lines were selected. The HIR delta E21 receptors were expressed on the cell surface, and they bound insulin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00212a021
更新日期:1993-12-14 00:00:00
abstract::Aureochromes have recently been shown to act as blue-light-regulated transcription factors in the stramenopile alga Vaucheria frigida. They comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module with flavin mononucleotide (FMN) as a chromophore and a basic region leucine zipper (bZIP) domain...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi400197u
更新日期:2013-05-07 00:00:00
abstract::The apolipoprotein apoE plays a key role in cholesterol and lipid metabolism. There are three isoforms of this protein, one of which, apoE4, is the major risk factor for Alzheimer's disease. At micromolar concentrations all lipid-free apoE isoforms exist primarily as monomers, dimers, and tetramers. However, the molec...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi1020106
更新日期:2011-04-05 00:00:00
abstract::A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0268706
更新日期:2003-01-14 00:00:00