Three constant fragments with different amino terminals, CL(105-214), CL(109-214), and CL(113-214), were obtained by limited proteolysis with trypsin or papain of a type lambda immunoglobulin light chain. The conformations of the three CL fragments were indistinguishable on the basis of circular dichroism and tryptophyl fluorescence spectra. The stability to heat and guanidine hydrochloride of CL(105-214) was almost the same as that of CL(109-214), but the stability of CL(113-214) was slightly lower than that of CL(105-214) or CL(109-214). The midpoint of the thermal unfolding transition at pH 7.5 was at 60.0 degrees C for CL(105-214), 60.4 degrees C for CL(109-214), and 57.5 degrees C for CL(113-214). The midpoint of the unfolding transition by guanidine hydrochloride at pH 7.5 and 25 degrees C was 1.2 M for CL(105-214) and CL(109-214) and at 1.0 M for CL(113-214). The kinetics of unfolding and refolding by guanidine hydrochloride of these CL fragments were analyzed on the basis of the three-species mechanism, U1 in equilibrium with U2 in equilibrium with N, where U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. It was found that only the microscopic unfolding rate constant for CL(113-214) is 2-3 times greater than that for CL(105-214) or CL(109-214) and that the other microscopic rate constants for the three CL fragments are all the same. These findings indicated that the amino-terminal residues, Gly-109-Lys-112, or a part of them, stabilize the CL(113-214) fragment by decreasing only the unfolding rate, that the transition state of the folding of the CL fragment is independent of the presence or absence of this peptide, and that, at the last step of folding, the peptide is incorporated into the globular domain, thus stabilizing it. Study holds ProTherm entries: 3962, 3963, 3964, 3965, 3966, 3968, 3969, 3970 Extra Details: amino-terminal residues; constant fragments;,microscopic rate constants; transition state
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:23 p.m.
|Number of data points||16|
|Proteins||Immunoglobulin kappa variable 1D-33 ; Immunoglobulin kappa variable 4-1|
|Assays/Quantities/Protocols||Experimental Assay: Cm buffers:Tris-HCl: 0.2 M, ionic:: ; Experimental Assay: dG_H2O buffers:Tris-HCl: 0.2 M, ionic:: ; Experimental Assay: Cm buffers:Sodium phosphate: 10 mM, ionic:NaCl: 0.15 M ; Experimental Assay: dG_H2O buffers:Sodium phosphate: 10 mM, ionic:NaCl: 0.15 M ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYSFGQGTKLEIKRTVAAPSVF|