Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry. The protein was found to contain a cysteinyl post-translational modification at Cys(214). Protein stability and conformation of MM-kappaI as a function of temperature or denaturant conditions at pH 7.4 and 4.8 were investigated. At pH 4.8, calorimetry demonstrated that MM-kappaI undergoes an incomplete, cooperative, partially reversible thermal unfolding with increased unfolding temperature and calorimetric enthalpy as compared to pH 7.4. Secondary and tertiary structural analyses provided evidence to support the presence of unfolding intermediates. Chemical denaturation resulted in more extensive protein unfolding. The stability of MM-kappaI was reduced and protein unfolding was irreversible at pH 4.8, thus suggesting that different pathways are utilized in thermal and chemical unfolding. Study holds ProTherm entries: 19220, 19221, 19222, 19223, 19224, 19225 Extra Details: hydrophobic core; beta-sheet; sequence analysis; pathways
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:51 p.m.
|Number of data points||13|
|Proteins||Immunoglobulin kappa variable 1D-33 ; Immunoglobulin kappa variable 4-1|
|Assays/Quantities/Protocols||Experimental Assay: Cm pH:4.8 ; Experimental Assay: Cm pH:7.4 ; Experimental Assay: dG_H2O ; Experimental Assay: Tm pH:4.8, prot_conc:0.37 mg/mL ; Experimental Assay: dHvH pH:4.8, prot_conc:0.37 mg/mL ; Experimental Assay: Tm pH:7.4, prot_conc:0.34 mg/mL ; Experimental Assay: dHvH pH:7.4, prot_conc:0.34 mg/mL ; Experimental Assay: dHcal pH:4.8 ; Experimental Assay: Tm prot_conc:1.73 mg/mL, pH:4.8 ; Experimental Assay: dHvH prot_conc:1.73 mg/mL, pH:4.8 ; Experimental Assay: dHcal pH:7.4 ; Experimental Assay: Tm prot_conc:1.73 mg/mL, pH:7.4 ; Experimental Assay: dHvH prot_conc:1.73 mg/mL, pH:7.4|
|Libraries||Mutations for sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYSFGQGTKLEIKRTVAAPSVF|