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

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

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

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

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

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

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

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

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

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

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

27 as being responsible for poor inhibition by BPTI.

28 thrombin, are both inhibited very slowly by BPTI.

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

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

31 or retention and degradation of destabilized BPTI variants.

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

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

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

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

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

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

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

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

40 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

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

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

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

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

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

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

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

48 35L BPTI mutant and the slowly unfolded G36D BPTI mutant exhibit low secretion efficiency.

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

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

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

52 The changes in BPTI and benzamidine affinity measure destabilization of

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

75 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein pr

76 0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been dete

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

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

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

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

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

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

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

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

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

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

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

88 ates of bovine pancreatic trypsin inhibitor (BPTI).

89 ants of bovine pancreatic trypsin inhibitor (BPTI).

90 orms of bovine pancreatic trypsin inhibitor (BPTI).

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

92 rotein, bovine pancreatic trypsin inhibitor (BPTI).

93 itin and basic pancreatic trypsin inhibitor (BPTI).

94 and the bovine pancreatic trypsin inhibitor (BPTI).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

114 that hydrophobic and polar residues modulate BPTI association.

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

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

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

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

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

120 This disulfide bond is found in native BPTI.

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

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

123 Nine BPTI variants with replacements that remove one or more

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

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

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

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

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

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

130 /- 26 nM) and showed that the enhancement of BPTI inhibition of fIXa by heparin was well described by

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

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

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

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

135 2, which constitute the binding interface of BPTI.

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

137 192Q-BPTI complex, the reactive site loop of BPTI is stabilized in a canonical conformation by severa

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

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

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

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

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

143 ing step in the oxidative folding pathway of BPTI.

144 nzyme and the amine leaving group portion of BPTI.

145 capability of mesotrypsin for proteolysis of BPTI and APPI.

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

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

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

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

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

151 conformation of the trypsin-binding site of BPTI.

152 retations regarding the aggregation state of BPTI in solution.

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

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

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

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

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

158 tom protein molecular dynamics trajectory of BPTI.

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

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

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

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

163 prOE17 to create the chimeric protein prOE17-BPTI.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

182 1H NMR spectroscopy of the BPTI (Lys15-Val) and BPTI (Lys15-Val, Pro13-Ile) mutants

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

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

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

186 5, P1 site) in a mode similar to that of the BPTI/trypsin interaction.

187 -38], that has recently been detected on the BPTI folding pathway.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

207 evidence of increased mobility compared with BPTI.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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