Abstract:
:The 77-amino acid pro-domain greatly accelerates the in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In this complex the pro-domain has a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helixes. When isolated from subtilisin, however, the pro-domain is 97% unfolded even under optimal folding conditions. The instability of the isolated pro-domain suggests that there may be a thermodynamic linkage between the stability of the pro-domain and its ability to facilitate subtilisin folding. On the basis of the X-ray crystal structure of the pro-domain subtilisin complex, we have designed stabilizing mutations in three areas of the pro-domain: alpha-helix 23-32 (E32Q), beta-strands 35-51 (Q40L), and alpha-helix 53-61 (K57E). These amino acid positions were selected because they do not contact subtilisin in the complex and because they appear to be in regions of the structure which are not well packed in the wild type pro-domain. Since none of the mutations directly contact subtilisin, their effects on the folding of subtilisin are linked to whether or not they stabilize a conformation of the pro-domain which promotes subtilisin folding. By sequentially introducing the three stabilizing mutations, the equilibrium for folding the pro-domain was shifted from 97% unfolded to 65% folded. By measuring the ability of these mutants to fold subtilisin, we are able to establish a correlation between the stability of the pro-domain and its ability to accelerate subtilisin folding. As the pro-domain is stabilized, the folding reaction becomes faster and distinctly biphasic. A detailed mechanism was determined for the double mutant, Q40L-K57E, which is 50% folded: P + Su if (30 800 M-1 s-1, 0.04 s-1) PSI if (0.07 s-1, <0.005 s-1) PS. PSI is an intermediate complex which accumulates in the course of the reaction, and PS is the fully folded complex. The more stable the pro-domain, the faster the folding reaction up to the point at which the isomerization of the intermediate into the fully folded complex becomes the rate-limiting step in the folding process.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Wang L,Ruan B,Ruvinov S,Bryan PNdoi
10.1021/bi972741rsubject
Has Abstractpub_date
1998-03-03 00:00:00pages
3165-71issue
9eissn
0006-2960issn
1520-4995pii
bi972741rjournal_volume
37pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00615a015
更新日期:1978-10-31 00:00:00
abstract::Thioltransferase activity was identified and the enzyme purified to apparent homogeneity from human red blood cells. Activity was measured as glutathione-dependent reduction of the prototype substrate hydroxyethyl disulfide; formation of oxidized glutathione (GSSG) was coupled to NADPH oxidation by GSSG reductase (1 u...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00239a002
更新日期:1991-06-25 00:00:00
abstract::The molecular chaperones Hsp90 and Hsp70 are highly regulated by various cochaperones that participate in the activation of steroid receptors. Here we study Tpr2 (also called DjC7), a TPR domain-containing type III J protein implicated in steroid receptor chaperoning. We propose that Tpr2 plays a role in the Hsp90-dep...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi800770g
更新日期:2008-08-05 00:00:00
abstract::Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochlor...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi015819j
更新日期:2002-03-19 00:00:00
abstract::The dual specificity mitogen-activated protein kinase phosphatase MKP3 downregulates mitogenic signaling through dephosphorylation of extracellular signal-regulated kinase (ERK). Like other MKPs, MKP3 consists of a noncatalytic N-terminal domain and a catalytic C-terminal domain. ERK binding to the N-terminal noncatal...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035346b
更新日期:2003-12-30 00:00:00
abstract::The membrane topology model of the Na(+)-citrate transporter CitS of Klebsiella pneumoniae shows a core of two homologous domains with opposite orientation in the membrane and each containing a so-called reentrant loop. A split version of CitS was constructed to study domain interactions and proximity relationships of...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi100336s
更新日期:2010-06-01 00:00:00
abstract::8-OxoGua (8-oxo-7,8-dihydroguanine) is produced in nucleic acids as well as in nucleotide pools of cells, by reactive oxygen species normally formed during cellular metabolic processes. MutT protein of Escherichia coli specifically degrades 8-oxoGua-containing deoxyribo- and ribonucleoside triphosphates to correspondi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi047550k
更新日期:2005-05-03 00:00:00
abstract::Genomic clones encompassing all the translated sequences, the 3' untranslated sequence, and 1 kb flanking the ATG translation initiation codon of bovine tropoelastin have been obtained and characterized by restriction enzyme analysis and extensive DNA sequencing. These analyses demonstrated that functionally distinct ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00432a003
更新日期:1989-03-21 00:00:00
abstract::Apolipoprotein AIV is one of the principal apolipoproteins synthesized by the rat small intestine. We have cloned a cDNA encoding a portion of preapolipoprotein AIV mRNA. A kinetically fractionated cDNA probe highly enriched for the abundant intestinal mucosal mRNA sequences was used to screen a library of recombinant...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00265a007
更新日期:1982-10-26 00:00:00
abstract::High-resolution, solid-state 15N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full 15N chemical shif...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00434a033
更新日期:1989-04-18 00:00:00
abstract::Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mecha...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi700225a
更新日期:2007-05-15 00:00:00
abstract::Many proteins utilize segmental motions to catalyze a specific reaction. The Omega loop of triosephosphate isomerase (TIM) is important for preventing the loss of the reactive enediol(ate) intermediate. The loop opens and closes even in the absence of the ligand, and the loop itself does not change conformation during...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049208d
更新日期:2004-09-14 00:00:00
abstract::Galactosyltransferase (UDPgalactose:glycoprotein galactosyltransferase EC 2.4.1.22) was isolated from swine mesentary lymhromatography on Sepharose 4B colums containing covalently bound p-aminophenyl-beta-D-N-acetylglucosamine. The homogenous enzyme showed a single band on disc gel electrophoresis and had a specific a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00668a009
更新日期:1976-11-16 00:00:00
abstract::The isolated N-terminal Src homology 3 (SH3) domain of Drosophila drk exists in equilibrium between folded and unfolded states in aqueous buffer near neutral pH. Nuclear magnetic resonance spectra recorded on both states simultaneously exhibit an approximate 1:1 ratio of protein conformations. The folded form is simil...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00020a025
更新日期:1995-05-23 00:00:00
abstract::We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0110140
更新日期:2001-10-30 00:00:00
abstract::HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue gr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi060278p
更新日期:2006-05-30 00:00:00
abstract::S100 beta is a calcium-binding protein, which regulates the activities of several enzymes and inhibits the phosphorylation of a variety of protein kinase C substrates in a calcium-dependent manner. The protein was recently found to activate a retinal membrane guanylate cyclase, and in this paper, we report that it inh...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9802115
更新日期:1998-07-28 00:00:00
abstract::The dynamic behavior of the rRNA A-site plays an important functional role. We have employed femtosecond time-resolved spectroscopy to investigate the nature of the conformational dynamics. In the drug-free state, the A-site samples multiple distinct conformations. Drug binding shifts the population distribution in a ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi400053s
更新日期:2013-03-12 00:00:00
abstract::The intact photosystem I of maize containing its full antenna complement (PSI-200) has been purified and fractionated into the core and outer antenna (LHCI) components. It is demonstrated by absorption and fluorescence spectroscopy that at least 80% of the long wavelength absorbing antenna pigments (red forms) are loc...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9813227
更新日期:1998-12-15 00:00:00
abstract::The interactions of Pr(III) and Eu(III) with specifically nitrated derivatives of the basic bovine pancreatic trypsin inhibitor have been studied using optical spectroscopy and nuclear magnetic resonance (NMR) at 250 and 270 MHz. Stability constants for proton and metal binding to nitrotyrosines 10 and 21 determined o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00623a015
更新日期:1977-02-22 00:00:00
abstract::Transition metal-transporting P1B-type CPx ATPases play crucial roles in mediating metal homeostasis and resistance in all cells. The degree to which N-terminal metal binding domains (MBDs) confer metal specificity to the transporter is unclear. We show that the two MBDs of the Zn/Cd/Pb effluxing pump Anabaena AztA ar...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi7006367
更新日期:2007-10-02 00:00:00
abstract::R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded enzyme that confers resistance to the antibacterial drug trimethoprim. This enzyme is not homologous in sequence or structure to chromosomal DHFRs. Equilibrium folding of tetrameric R67 DHFR was studied and found to be fully reversible. Formation of an inactiv...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00180a018
更新日期:1994-04-12 00:00:00
abstract::NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of G(B1) which is on the margin of stability. At 5 ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9920230
更新日期:2000-02-08 00:00:00
abstract::The dependence of enzymatic catalysis on diffusion rates in solution was examined with regard to high specific activity carbonic anhydrase (CA II) by varying the viscosity of the reaction medium with added glycerol, sucrose, and ficoll (a copolymer of sucrose and epichlorohydrin). Responses of the Michaelis-Menten par...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00383a028
更新日期:1987-05-05 00:00:00
abstract::Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi701448p
更新日期:2008-01-08 00:00:00
abstract::We have used diethyl pyrocarbonate (DEP), which carbethoxylates adenine bases, and dimethyl sulfate (DMS), which methylates guanine residues and single-stranded cytosines, to probe bases in open complexes between RNA polymerase and the lac UV5 promoter in vitro. We compared the kinetics of reactivity between bases in ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00436a040
更新日期:1989-05-16 00:00:00
abstract::N1-(5'-Phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (HisI, PR-AMP cyclohydrolase) is a central enzyme in histidine biosynthesis catalyzing the hydrolysis of the N1-C6 bond of the purine substrate, a reaction unique to this pathway. A source of the recombinant monofunctional Methanococcus vannielii PR-AMP c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi982475x
更新日期:1999-02-02 00:00:00
abstract::The solution structure of the C-terminal fragment 255-316 of thermolysin has been determined by two-dimensional proton NMR methods. For this disulfide-free fragment there was a previous proposal according to which it would fold into a stable helical structure forming a dimer at concentrations above 0.06 mM. A complete...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00253a023
更新日期:1994-12-13 00:00:00
abstract::The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and char...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049711q
更新日期:2004-05-25 00:00:00
abstract::A cross-linked tripeptide has been isolated from alkaline hydrolysates of NaB3H4-reduced calf bone collagen. The peptide contains dihydroxylysinonorleucine, the most abundant cross-link in bone collagen, and it has a single N-terminal proline and a single C-terminal valine. These amino acids are in peptide linkage wit...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00691a011
更新日期:1975-10-07 00:00:00