Independent folding and conformational changes of the barnase module in the VL-barnase immunofusion: calorimetric evidence

• Published in: FEBS letters Vol. 557; no. 1; pp. 248 - 252
• Main Authors: Tsybovsky, Yaroslav I, Kedrov, Alexey A, Martsev, Sergey P
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 16.01.2004
• Subjects: Antiferritin; Bacillus - enzymology; Bacillus amyloliquefaciens; Bacterial Proteins; Calorimetry, Differential Scanning; Cloning, Molecular; Differential scanning calorimetry; Escherichia coli - enzymology; Ferritins - immunology; GMP, guanosine 3′-monophosphate; Humans; Immunotoxin; Protein Conformation; Protein Folding; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Ribonucleases - chemistry; Ribonucleases - genetics; Ribonucleases - metabolism; RNase; scFv, single-chain Fv fragment; Thermodynamics; VH, antibody heavy chain variable domain; VL, antibody light chain variable domain; Differential scanning calorimetry; Immunotoxin; VL, antibody light chain variable domain; scFv, single-chain Fv fragment; VH, antibody heavy chain variable domain; Protein folding; Antiferritin; GMP, guanosine 3′-monophosphate; RNase
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(03)01509-6

Although stability is critical for in vivo application of immunotoxins, a thermodynamic description of their folding/stability is still lacking. We applied differential scanning calorimetry (DSC) to RNase-based immunofusion comprising barnase, cytotoxic RNase from Bacillus amyloliquefaciens, fused to the light chain variable domain (VL) of anti-human ferritin antibody F11. By analyzing DSC curves recorded with or without preheating and addition of the barnase-stabilizing ligand guanosine 3′-monophosphate, we (i) assigned two well-resolved thermal transitions to the VL and barnase modules of VL-barnase, (ii) demonstrated independent folding of these two modules, and (iii) showed altered stability of the barnase module, which resulted from the dimeric state of VL-barnase.