Chemical Synthesis, Molecular Cloning, Overexpression, and Site-Directed Mutagenesis of the Gene Coding for Pumpkin (Curcubita maxima) Trypsin Inhibitor CMTI-V

Wen, Lisa; Kim, S.S.; Tinn, T.T.; Huang, Jenq‐Kuen; Krishnamoorthi, Ramaswamy; Gong, Yuping; Lwin, Y. N.; Kyin, Saw

doi:10.1006/prep.1993.1028

Cited by 10 publications

(16 citation statements)

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“…Mutations were confirmed by DNA sequencing. The proteins were purified using a modified procedure of Wen et al (1993): the pH of the cell-free extract was adjusted to 3.5, and precipitated impurities were removed by centrifugation. The sample was further purified by ultrafiltration, followed by reverse-phase HPLC, and characterized by trypsin-inhibition assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

Section: Proteinsmentioning

confidence: 99%

Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V

et al. 2001

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Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T m ), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (⌬⌬G d 50°C ‫ס‬ −4 kcal/mole; ⌬T m ‫ס‬ −22°C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (⌬⌬G d 50°C ‫ס‬ −3 kcal/mole; ⌬T m ‫ס‬ −11°C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys 44 -Asp 45 peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (⌬⌬G d 50°C ‫ס‬ −1 kcal/mole; ⌬T m ‫ס‬ +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (⌬⌬G d 50°C ‫ס‬ +1 kcal/mole; ⌬T m ‫ס‬ +17°C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.Keywords: Disulfide-bond; cross-link; protein stability; differential scanning calorimetry; denaturation; folding Supplemental material: See www.proteinscience.org.The conformational stability of a protein is important to its function. Certain diseases such as Alzheimer's, prion, and cystic fibrosis, are associated with misfolded, unfolded, or aggregated proteins (Kelly 1996;Harper and Lansbury, Jr. 1997;Horwich and Weissman 1997, Qu et al. 1997). Much has been characterized regarding contributions to protein stability of hydrogen bonds, i...

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Section: Proteinsmentioning

confidence: 99%

Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V

et al. 2001

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“…The "N-labeled recombinant CMTI-111 was cleaved off GST by digestion with thrombin and purified by reverse-phase HPLC. The recombinant protein (yield = 3-5 mg/L of cell culture), rCMTI-111, was found to inhibit trypsin by the assay method described previously (Wen et al, 1993). The nucleic acid and amino acid sequences of rCMTI-111 are as follows:…”

Section: "N-labeled Recombinant Cmti-111mentioning

confidence: 99%

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family

et al. 1998

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Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49593‐626), whereby an enzyme‐catalyzed equilibrium between intact (I) and reactive‐site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI‐III; Mr 3 kDa) of the squash family and rCMTI‐V (Mr ∼ 7 kDa) of the potato I family. These two inhibitors have different binding loop‐scaffold interactions and different Khyd values—2.4 (CMTI‐III) and 9 (CMTI‐V)—at 25°C. The reactive‐site peptide bond (P1‐P'1) is that between Arg5 and Ile6 in CMTI‐III, and that between Lys44 and Asp45 in CMTI‐V. The order parameters (S2) of backbone NHs of uniformly 15N‐labeled rCMTI‐III and rCMTI‐III* were determined from measurements of 15N spin‐lattice and spin‐spin relaxation rates, and {1H}‐15N steady‐state heteronuclear Overhauser effects, using the model‐free formalism, and compared with the data reported previously for rCMTI‐V and rCMTI‐V*. The backbones of rCMTI‐III (〈S2〉 = 0.71) and rCMTI‐III* (〈S2) = 0.63) are more flexible than those of rCMTI‐V (〈S2〉 = 0.83) and rCMTI‐V* (〈S2) = 0.85). The binding loop residues, P4‐P1, in the two proteins show the following average order parameters: 0.57 (rCMTI‐III) and 0.44 (rCMTI‐III*); 0.70 (rCMTI‐V) and 0.40 (rCMTI‐V*). The P1′‐P4′ residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI‐III) and 0.47 (rCMTI‐III*); and 0.73 (rCMTI‐V) and 0.83 (rCMTI‐V*). The newly formed C‐terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI‐III* due to the Cys3‐Cys20 crosslink. In contrast, the newly formed N‐terminal (Pn′ residues) becomes more flexible only in rCMTI‐III*, most likely due to lack of an interaction between the P1′ residue and the scaffold in rCMTI‐III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn′ residues seem to facilitate the resynthesis of the P1‐P1′ bond, leading to a lower Khyd value.

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“…In fact, low molecular weight substrates for P-Factor Xlla have been shown to be even better substrates for kallikrein (Hojima et al, 1980;McRae et al, 1981). Wen et al (1993) designed a chemically-synthesized DNA based on the amino add sequence of CMTI-V, and expressed it as a fusion protein (Table 4). Longstaff et aL (1990) and Heinz et al (1992) also altered the specificity of Eglin c through substitutions at PI.…”

Section: Dissodation Constants Of Cmti-v In Complex With Trypsin and mentioning

confidence: 99%

“…Mutant forms of CMTI-V have been studied (Wen, 1993;Wen, 1995), focusing on the PI, P2, P3', P6', and P8' positions, EUid removal of the disulfide bridge. The wild-type P2 is Thr, which is involved in hydrogen bonding interactions with other loop residues.…”

Section: Dissodation Constants Of Cmti-v In Complex With Trypsin and mentioning

confidence: 99%

“…Site-spedfic mutations were made in the internal sequences of Clone 70-1, which is a fusion protein based in tJie wildtj^pe's amino add sequence (Wen, 1993), hereafter referred to as wildtype. The mutant genes were expressed and activity of their mutant protein products were compared with wild-type CMTI-V activity.…”

Section: Serine Proteinase/inhibitor Complex and Mutant Selectionmentioning

confidence: 99%

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Structure-function study of a plant protein proteinase inhibitor: site-directed mutagenesis of Cucurbita maxima trypsin inhibitor V

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Chemical Synthesis, Molecular Cloning, Overexpression, and Site-Directed Mutagenesis of the Gene Coding for Pumpkin (Curcubita maxima) Trypsin Inhibitor CMTI-V

Cited by 10 publications

References 0 publications

Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V

Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family

Structure-function study of a plant protein proteinase inhibitor: site-directed mutagenesis of Cucurbita maxima trypsin inhibitor V

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