Independent folding and conformational changes of the barnase module in the VL-barnase immunofusion: calorimetric evidence.
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.
Tsybovsky
Yaroslav I
YI
Institute of Bio-Organic Chemistry, National Academy of Sciences of Belarus, Minsk 220141, Belarus.
Kedrov
Alexey A
AA
Martsev
Sergey P
SP
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005293:Ferritins / Q000276:immunology; D006801:Humans; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005293:Ferritins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease