Differential modulation of binding loop flexibility and stability by Arg super(50) and Arg super(52) in Cucurbita maxima trypsin inhibitor-V deduced by trypsin-catalyzed hydrolysis and NMR spectroscopy

• Published in: Biochemistry (Easton) Vol. 35; no. 15; pp. 4784 - 4794
• Main Authors: Cai, Mengli, Huang, Ying, Prakash, O, Wen, L, Dunkelbarger, S P, Huang, Jeng-Kuen, Lui, Jianhua, Krishnamoorthi, R
• Format: Journal Article
• Language: English
• Published: 01.01.1996
• Subjects: Cucurbita maxima
• ISSN: 0006-2960

The side chains of Arg super(50) and Arg super(52) in Cucurbita maxima trypsin inhibitor-V (CMTI-V) anchor the binding loop to the scaffold region. The consequences of these hydrogen-bonding and electrostatic interactions on the conformational flexibility and stability of the binding loop were evaluated by trypsin-catalyzed hydroplysis of CMTI-V mutants, in which each of the two arginines was individually replaced with Ala, Lys, or Gln by genetic engineering methods. All the mutants exhibited significantly increased vulnerability to the protease attack at many sites, including the reactive-site (Lys super(44)-Asp super(45) peptide bond), with the R50 mutants showing much more pronounced effects than the R52 counterparts. For CMTI-V and the mutants studied, a qualitative correlation was inferred between binding loop flexibility and retention time on reverse-phase high-pressure liquid chromatography C-18 column. The R50 mutants were found to be more flexible than the corresponding R52 versions. These results demonstrate the Arg super(50) contributes more to the stability and function of CMTI-V. The differing strengths of the hydrogen bonds made by Arg super(50) and Arg super(52) were characterized by determining the internal dynamics of their side chains at pH 5.0 and 2.5: super(15) N NMR longitudinal and transverse relaxation rates and super(15)N- super(1)H nuclear Overhauser effect (NOE) enhancements were measured for the main-chain and side-chain NH groups in super(1)5N-labeled recombinant CMTI-V (rCMTI-V) and the model-free parameters [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559; 4559-4570] were calculated. At both pH 5.0 and 2.5, the arginines at positions 26, 47, 58, and 66 are found to be highly mobile, as the calculated general order parameters. S super(2) values, of their N epsilon H groups fall in the range 0.03-0.18. The corresponding values for Arg super(50) and Arg super(52) are 0.73 and 0.63, respectively, at pH 5.0, thus confirming that the two arginines are rigid and hyydrogen-bonded. At pH 2.5, these hydrogen bonds are still retained with Arg super(50) appearing to be more restrained (S super(2) = 0.71) than Arg super(52) (S super(2) = 0.56). This is consistent with a greater contribution by Arg super(50) to the conformational stability of the reactive-site loop in CMTI-V. The results also indicate that the and Arg super(50) and Arg super(52) side chains are not hydrogen-bonded to carboxylate groups, which would be protonated at pH 2.5 and, hence, unavailable for hydrogen-bonding interactions. The overall folding of rCMTI-V appears not to be significantly affected by the pH change, as indicated by comparison of super(1)H and super(15)N chemical shifts, sequential NOE cross-peaks, and S super(2) values of the backbone atoms, and the conserved side-chain dynamics of Trp super(9) and Trp super(54) - residues that are involved in hydrophobic and hydrogen-bonding interactions with others in the protein core and the binding loop, respectively.