ライフサイエンスコーパス: BPTI

1 BPTI and hirudin exemplify two extreme cases of such div

2 BPTI and OI are members of the Kunitz and Kazal families

3 BPTI containing only a single disulfide bond (5-55) is n

4 interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skele

5 A BPTI mutant containing 22 alanine residues was further s

6 tails of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes.

7 ible single and pairwise cysteine to alanine BPTI mutants, and the effect of these mutations on secre

8 n the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds b

9 Such mutants have higher activity and BPTI affinity than trypsinogen, which indicates that the

10 ding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues t

11 ral differences between the dendrotoxins and BPTI at the anti-protease loop; this explains the inabil

12 exhibited little or no change in heparin and BPTI binding or in antimicrobial function.

13 udies of the interaction between heparin and BPTI indicated an unstable interaction with very low aff

14 ion exists between the values of kcat/Km and BPTI affinity of mutant trypsinogens and trypsins.

15 glycerate kinase, hen egg-white lysozyme and BPTI, conformational heterogeneity arises from a number

16 sed as central to the binding of trypsin and BPTI and protein complex formation in general.

17 ions because opposite charges in trypsin and BPTI draw them together.

18 Using ubiquitin and BPTI as examples, we demonstrate how the approach allows

19 NMR spectroscopy of the BPTI (Lys15-Val) and BPTI (Lys15-Val, Pro13-Ile) mutants indicates that small

20 Because BPTI binding was comparable in wild-type and Loop 3/Loop

21 Thrombin and the E192Q mutant, which binds BPTI much more tightly than thrombin, are both inhibited

22 Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structur

23 active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the per

24 mediated folding/unfolding reaction of BPTI, BPTI variants based on a native-like two-disulfide analo

25 dence suggests that the inhibition of HLE by BPTI homologues probably takes place by a two-step mecha

26 of thrombin or thrombin E192Q inhibition by BPTI approximately 10-fold.

27 he TM effect on thrombin E192Q inhibition by BPTI is primarily on the first, reversible step in the r

28 kinetics and thermodynamics of inhibition by BPTI toward trypsin and chymotrypsin were investigated.

29 as being responsible for poor inhibition by BPTI.

30 thrombin, are both inhibited very slowly by BPTI.

31 retion of BPTI in an intact eucaryotic cell, BPTI was expressed and secreted from a synthetic gene in

32 We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that

33 or retention and degradation of destabilized BPTI variants.

34 An additional mutation at P3, i.e., BPTI (Lys15-Val, Pro13-Ile), increased the inhibition of

35 Structural studies of the thrombin E192Q-BPTI complex have previously shown that the 60 loop lies

36 the crystal structure of the thrombin E192Q-BPTI complex, the reactive site loop of BPTI is stabiliz

37 arities of folding mechanism(s) between EGF, BPTI, and hirudin are discussed in this paper.

38 Partially folded BPTI undergoes local motions that are slow, noncooperati

39 s demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envel

40 s suggest that a significant flux of folding BPTI molecules proceed through the one-disulfide interme

41 ate buffer and 9.2 x10(-4) cm(3)mol/g(2) for BPTI in MOPS.

42 sphate buffer, 1.6 x10(-4) cm(3)mol/g(2) for BPTI in phosphate buffer and 9.2 x10(-4) cm(3)mol/g(2) f

43 time scale at ambient T, while our data for BPTI and activation parameters available for ring-flippi

44 ic residues, is feasible as an interface for BPTI self-association.

45 nformation-coupled redox folding pathway for BPTI(Ala14)Ala38 involving two parallel paths with unfol

46 In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pr

47 porting the notion that the binding site for BPTI is distinct from the site involved in heparin bindi

48 ngineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stab

49 native protein structure could be built from BPTI sequences that contained many alanine residues dist

50 35L BPTI mutant and the slowly unfolded G36D BPTI mutant exhibit low secretion efficiency.

51 mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI.

52 ins of Cys 14, Lys 15, Cys 38, and Arg 39 in BPTI.

53 h of the fifteen possible disulfide bonds in BPTI, starting from the reduced, unfolded protein.

54 The changes in BPTI and benzamidine affinity measure destabilization of

55 stitution for this buried 30-51 disulfide in BPTI.

56 erent "active" conformations are involved in BPTI binding and substrate hydrolysis.

57 eveal that the key interactions (observed in BPTI-trypsin complex) needed for anti-protease activity

58 form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with

59 ures of bovine pancreatic trypsin inhibitor (BPTI) and alpha-DtX.

60 tion by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhi

61 nted by bovine pancreatic trypsin inhibitor (BPTI) and hirudin.

62 ies, in bovine pancreatic trypsin inhibitor (BPTI) and in the I76A mutant of barnase, represent very

63 face of bovine pancreatic trypsin inhibitor (BPTI) and surrounding residues were substituted individu

64 bles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by

65 scale in basic pancreatic trypsin inhibitor (BPTI) are investigated using nuclear magnetic resonance

66 nts' in bovine pancreatic trypsin inhibitor (BPTI) are the two long strands of antiparallel beta-shee

67 hibitor bovine pancreatic trypsin inhibitor (BPTI) as a model system.

68 Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respect

69 reduced bovine pancreatic trypsin inhibitor (BPTI) at high temperature in water and a control simulat

70 n E192Q-bovine pancreatic trypsin inhibitor (BPTI) complex, the structural basis for the slow reactiv

71 face of bovine pancreatic trypsin inhibitor (BPTI) destabilizes the protein by approximately 5 kcal/m

72 Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of pro

73 Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant c

74 Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a model protein to investi

75 ment in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance

76 ding of bovine pancreatic trypsin inhibitor (BPTI) has served as a paradigm for the folding of disulf

77 ants of bovine pancreatic trypsin inhibitor (BPTI) in yeast.

78 hway of bovine pancreatic trypsin inhibitor (BPTI) is characterized by the predominance of folding in

79 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein pr

80 0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been dete

81 to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural as

82 ield of bovine pancreatic trypsin inhibitor (BPTI) severalfold, an effect that was enhanced when redu

83 hibitor bovine pancreatic trypsin inhibitor (BPTI) to probe fIXa reactivity in the absence and in the

84 ries of bovine pancreatic trypsin inhibitor (BPTI) variants with similar stabilities and structures h

85 mics of bovine pancreatic trypsin inhibitor (BPTI) were examined using 15N NMR relaxation experiments

86 gues of bovine pancreatic trypsin inhibitor (BPTI) with the proteolytically inactive rat trypsin muta

87 iant of bovine pancreatic trypsin inhibitor (BPTI), [14-38]Abu, is a partially folded ensemble which

88 nd that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin

89 ite for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteina

90 The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activit

91 iate of bovine pancreatic trypsin inhibitor (BPTI), with the disulfide between Cys14 and Cys38 reduce

92 ates of bovine pancreatic trypsin inhibitor (BPTI).

93 ants of bovine pancreatic trypsin inhibitor (BPTI).

94 orms of bovine pancreatic trypsin inhibitor (BPTI).

95 d 51 in bovine pancreatic trypsin inhibitor (BPTI).

96 rotein, bovine pancreatic trypsin inhibitor (BPTI).

97 itin and basic pancreatic trypsin inhibitor (BPTI).

98 and the bovine pancreatic trypsin inhibitor (BPTI).

99 bitors: bovine pancreatic trypsin inhibitor (BPTI, KD = 7.0 microM for Bslo and 2.6 microM for Dslo)

100 cted on bovine pancreatic trypsin inhibitor (BPTI; 58 residues) suggested that if cumulative mutation

101 creasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecula

102 Interestingly, BPTI folds more efficiently in the presence of 5 mM GSSG

103 e two-disulfide analog of this intermediate, BPTI(Ala14)Ala38, were examined.

104 The amino acid replacements introduced into BPTI(Ala14)Ala38 rendered it thermodynamically less stab

105 aI16V17 trypsinogen is the lone outlier; its BPTI affinity is higher than would be expected based on

106 tures that are not found in classical Kunitz/BPTI proteins and suggest the mode of interaction with t

107 lphide bridges not found in classical Kunitz/BPTI proteins.

108 us has a sequence resembling those of Kunitz/BPTI proteins.

109 ick salivary gland product related to Kunitz/BPTI proteins is a potent inhibitor of human beta-trypta

110 At the single-channel level, BPTI and OI were found to inhibit KCa channels by a simi

111 into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their targ

112 t with studies of other model proteins, like BPTI, in which formation of a single disulfide bond is s

113 nondenaturing concentrations of urea (2 M), BPTI behaves as a monomer, suggesting that hydrophobic a

114 ed to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric

115 ell as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes.

116 Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostati

117 At 130 mg/mL BPTI, however, the fractal dimension was not significant

118 that hydrophobic and polar residues modulate BPTI association.

119 bands predicted for the wild-type and mutant BPTI have much less intensity than observed experimental

120 various extents for the wild-type and mutant BPTI.

121 vitro folding or unfolding rates, but mutant BPTI secretion is directly correlated with the in vitro

122 t ordered residues are those that, in native BPTI, fold into the slow exchange core.

123 In native BPTI, there is an unusual polar interaction between the

124 This disulfide bond is found in native BPTI.

125 proteins, the overall conformation of native BPTI was retained, and the relaxation data for these pro

126 o a structure very similar to that of native BPTI, and to be a functional trypsin inhibitor.

127 Nine BPTI variants with replacements that remove one or more

128 cattering profile observed in the absence of BPTI closely matched that predicted for an ensemble of r

129 residues are not involved in the binding of BPTI to azurocidin, supporting the notion that the bindi

130 onded to the P2 and P4 backbone carbonyls of BPTI).

131 n, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-B

132 The stable complex of BPTI and trypsin was inactive as a KCa channel inhibitor

133 election, binding-competent conformations of BPTI and trypsin are already present in the apo ensemble

134 related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for t

135 /- 26 nM) and showed that the enhancement of BPTI inhibition of fIXa by heparin was well described by

136 conclude that partially folded ensembles of BPTI, even those with little or no CD- or NMR-detectable

137 icture of the early events in the folding of BPTI, our results address quantitatively the effect of l

138 uggested to be a main site for initiation of BPTI folding.

139 Tyr23 and Ala25, is crucial to initiation of BPTI folding.

140 2, which constitute the binding interface of BPTI.

141 on of two-disulfide folding intermediates of BPTI and thus did not appear to appreciably catalyze the

142 192Q-BPTI complex, the reactive site loop of BPTI is stabilized in a canonical conformation by severa

143 the idea that the trypsin inhibitory loop of BPTI recognizes a specific site on the channel protein.

144 Lys15 is located on a loop of BPTI that forms the primary contact region for binding t

145 ility for a series of six point mutations of BPTI.

146 in the disulfide-coupled folding pathway of BPTI because of its participation in the rate-determinin

147 no glutathione (GSH), the folding pathway of BPTI proceeds through a nonproductive route via N* (a tw

148 ing step in the oxidative folding pathway of BPTI.

149 nzyme and the amine leaving group portion of BPTI.

150 capability of mesotrypsin for proteolysis of BPTI and APPI.

151 We show that the rate of BPTI association is slower for DeltaI16V17 trypsinogen t

152 ation revealed that the dissociation rate of BPTI from the bovine KCa channel is fast (k(off) = 0.41

153 lfide-mediated folding/unfolding reaction of BPTI, BPTI variants based on a native-like two-disulfide

154 s in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expressed an

155 ng affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes

156 conformation of the trypsin-binding site of BPTI.

157 retations regarding the aggregation state of BPTI in solution.

158 culated for the form II crystal structure of BPTI showed the best agreement with experiment.

159 ne, were used to manipulate the structure of BPTI.

160 eir folding mechanism(s) differ from that of BPTI by 1) a higher degree of heterogeneity of 1- and 2-

161 cribed by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenc

162 s a pathway conspicuously similar to that of BPTI, exhibiting limited species of folding intermediate

163 tom protein molecular dynamics trajectory of BPTI.

164 istics of [14-38]Abu, a synthetic variant of BPTI that is partially folded in aqueous buffer near neu

165 Using recombinant variants of BPTI, we have determined the rate constants correspondin

166 In particular, BPTI's P1 residue adapts to the S1 pocket and prime site

167 the different cavities, with only the polar BPTI cavity predicted to be hydrated.

168 Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstra

169 prOE17 to create the chimeric protein prOE17-BPTI.

170 millisecond simulation of the folded protein BPTI reveals a small number of structurally distinct con

171 nyl and tyrosinyl rings of the 6 kDa protein BPTI have been investigated by NMR at temperatures betwe

172 nt millisecond-long MD trajectory of protein BPTI is employed to simulate the time variation of amide

173 molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides tha

174 oth past experiments suggesting that reduced BPTI is a random coil and more recent experiments provid

175 hed ER-associated degradation do not secrete BPTI more efficiently, indicating that retention and deg

176 than for a mutant trypsinogen with a similar BPTI affinity.

177 his hypothesis, we designed and produced six BPTI mutants containing from 21 to 29 alanine residues.

178 rmolecular interactions, as observed in some BPTI crystal structures, without the formation of stable

179 ovement at the apex of the 60 loop, and that BPTI is found in the same canonical orientation as in th

180 ubiquitin, diffusion constants indicate that BPTI dimerizes at concentrations above about 3 mg/mL and

181 This observation suggests that BPTI binds to an "active" trypsinogen conformation that

182 The BPTI analogue termed [14-38](Abu) retains only the disul

183 The BPTI surface shows that while one side is highly charged

184 may play a distinctive role in defining the BPTI folding mechanism.

185 out" the buried 30-51 disulfide bond in the BPTI molecule.

186 The mutation of Lys15 to Ala increases the BPTI affinity and activity of trypsinogen to an even gre

187 mutation of Asp194 to Asn also increases the BPTI affinity and activity of trypsinogen.

188 1H NMR spectroscopy of the BPTI (Lys15-Val) and BPTI (Lys15-Val, Pro13-Ile) mutants

189 The majority of the BPTI mutants exhibited a binding affinity similar to tha

190 to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15

191 the stability, folding, and dynamics of the BPTI.

192 5, P1 site) in a mode similar to that of the BPTI/trypsin interaction.

193 -38], that has recently been detected on the BPTI folding pathway.

194 viously shown that the 60 loop lies over the BPTI, a position which requires 8 A movement at the apex

195 Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-

196 alytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site

197 y dithiothreitol, of the disulfides in these BPTI(Ala14)Ala38 variants was also decreased by the subs

198 The interactions of three BPTI homologues with human leukocyte elastase and porcin

199 gree of structure detected among these three BPTIs in solution by several biophysical techniques, the

200 mesotrypsin-BPTI and human cationic trypsin-BPTI complexes.

201 Correctly folded, wild type BPTI contains a disulfide at the 30-51 positions, with t

202 lar to that of the native state of wild type BPTI, although they are severely destabilized relative t

203 ulfide (30-51) intermediate in the wild-type BPTI refolding reaction.

204 new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62

205 complex between S195A trypsin and wild-type BPTI was also solved.

206 are secreted at half the level of wild-type BPTI, while secretion of the Cys51 mutant is reduced by

207 ed [14-38]Abu and fully reduced and unfolded BPTI analogue were determined from heteronuclear NMR rel

208 are implied by NOEs in reduced and unfolded BPTI, between residues Tyr23 and Ala25, and between Gly3

209 tive-like structure detected in the unfolded BPTI is important in folding.

210 g contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfold

211 ductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that

212 With BPTI added to a concentration of 65 mg/mL, there was a c

213 evidence of increased mobility compared with BPTI.

214 tructures of trypsin mutants in complex with BPTI suggest that these four residues function cooperati

215 conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but a

216 a conformation that favors interactions with BPTI, probably involving motion of the 60 loop.

217 iation measurements have been performed with BPTI under a variety of temperature, pH, salt, urea cond

218 While fIXa alone was poorly reactive with BPTI (K(i) approximately 0.7 mM), this reactivity was in

219 In wild-type (WT) BPTI, Gly 37 HN is in an unusual NH-aromatic-NH network

220 ss favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor.

221 ggest that the slow internal motions in Y35G BPTI are more independent in the absence of the 14-38 di

222 type protein, reducing the disulfide in Y35G BPTI significantly decreased the number of backbone amid

223 of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography

224 a wider range than those seen in native Y35G BPTI.

225 The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolu

226 ies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein.

227 that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of

228 vitro is found; both the rapidly folded Y35L BPTI mutant and the slowly unfolded G36D BPTI mutant exh