title: Differential modulation of binding loop flexibility and stability by Arg50 and Arg52 in Cucurbita maxima trypsin inhibitor-V deduced by trypsin-catalyzed hydrolysis and NMR spectroscopy.
abstract: The side chains of Arg50 and Arg52 iin Cucurbita maxima trypsin inhibitor-V (CMTI-V) anchor the binding loop to the scaffold region [Cai, M., Gong, Y., Kao, J.L-F., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The consequences of these hydrogen-bonding and electrostatic interactions on the conformational flexibility and stability of the binding loop were evaluated by trypsin-catalyzed hydrolysis of CMTI-V mutants, in which each of the arginines was individually replaced with Ala, Lys, or Gln by genetic engineering methods. All mutants exhibited significantly increased vulnerability to the protease attack at many sites, including the reactive-site (Lys44-Asp45 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 a 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 that Arg50 contributes more to the stability and function of CMTI-V. The differing strengths of the hydrogen bonds made by Arg50 and Arg52 were characterized by determining the internal dynamics of their side chains at pH 5.0 and 2.5: 15N NMR longitudinal and transverse relaxation rates and 15N-1H nuclear Overhauser effect (NOE) enhancements were measured for the main-chain and side-chain NH groups in 15N-labeled recombinant CMTI-V (rCMTI-V) and the model-free parameters [Lipari, G., & Szabo, A.(1982) J. Am. Chem. Soc. 104, 4546-59; 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 caluculated general order parameters, S2 values, of their NepsilonH groups fall in the range 0.03-0.18. The corresponding values for Arg50 amd Arg52 are 0.73 and 0.63, respectively, at pH 5.0, thus confirming that the two arginines are rigid and hydrogen bonded. At pH 2.5, these hydrogen bonds are still retained with Arg50 appearing to be more restrained (S2 = 0.71) than Arg52 (S2 = 0.56). This is consistent a greater contribution by Arg50 to the conformational stability of the reactive-site loop in CMTI-V. The results also indicate that the Arg50 and Arg52 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 comparisons of 1H and 15N chemical shifts, sequential NOE cross-peaks, and S2 values of the backbone atoms, and the conserved side-chain dynamics of Trp9 and Trp54--residues that are involved in hydrophobic and hydrogen-bonding interactions with others in the protein core and the binding loop, respectively.
authors.affiliation: Departmemt of Biochemistry, Kansas State University, Manhattan, 66506, USA.
authors.forename: M
authors.initials: M
authors.lastname: Cai
authors.forename: Y
authors.initials: Y
authors.lastname: Huang
authors.forename: O
authors.initials: O
authors.lastname: Prakash
authors.forename: L
authors.initials: L
authors.lastname: Wen
authors.forename: S P
authors.initials: SP
authors.lastname: Dunkelbarger
authors.forename: J K
authors.initials: JK
authors.lastname: Huang
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: R
authors.initials: R
authors.lastname: Krishnamoorthi
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry*
chemical_list: C081870:CMTI protein, Cucurbita maxima; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D001120:Arginine