Cold-induced conformational changes of ribonuclease A as investigated by subzero transverse temperature gradient gel electrophoresis.
Subzero temperature gradient gel electrophoresis is a new approach which allows to measure the transition temperature of low temperature-induced subtle conformational changes of proteins and to detect the different conformational states, including unfolded states. Using this technique under destabilizing conditions, i.e., in the presence of 4 M urea, bovine pancreas ribonuclease A exhibited two transitions: (i) a continuous transition with a midpoint temperature of -14 degrees C corresponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discontinuous transition at -22.5 degrees C from intermediate to a non migrating species. Under reducing conditions this second transition was shifted toward high temperatures (-18.5 degrees C). We attempted to detect these two transitions by differential scanning calorimetry, UV spectrophotometry and circular dichroism measurements. These transitions have been ascribed to subtle cold-induced conformational changes.
Curtil
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C
Centre de Recherches du Service de Santé des Armées, Unité de Biochimie, La Tronche, France.
Channac
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L
Ebel
C
C
Masson
P
P
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011754:Pyronine / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D014508:Urea
D014508:Urea; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012259:Ribonuclease, Pancreatic; D011754:Pyronine