Structural and functional changes associated with modification of the ubiquitin methionine.
The effects of oxidation and cleavage of Met-1 of ubiquitin on conformation and biological activity were individually investigated. Proton NMR studies demonstrated that oxidation to the sulfone led to restricted structural perturbations at neutral pH, particularly in the vicinity of Ile-61. Below pH 3, in the presence of acetic acid, oxidation to the sulfone facilitated a conformational expansion demonstrable by retardation on gel electrophoresis and CD changes below 210 nm. The predominant phase of the low-pH transition did not involve significant changes in alpha-helix content, indicating the capacity of ubiquitin for limited structural transitions. Cleavage of Met-1 by CNBr, on the other hand, was associated with a global unfolding transition below pH 4 that involved a major loss of alpha-helix. Differences in the behavior of the native and des-Met proteins at low pH indicate that Met-1 contributes a minimum of 3.4 kcal/mol to the stability of the native conformation. Two Met-1 sulfoxide isomers, of markedly different conformational stability, were formed by treatment of ubiquitin with H2O2. One isomer was similar in stability to the sulfone, while the other was intermediate in stability between the sulfone and des-Met proteins, the differences potentially interpretable in terms of the geometry of the Met-1-Lys-63 hydrogen bond. The overall activities of the oxidized and des-Met derivatives in ATP-dependent proteolysis differed subtly from that of native ubiquitin. The unresolved sulfoxides exhibited an approximately 50% increase in activity, while the sulfone and des-Met proteins exhibited a 50% decrease in activity at low concentrations and normal activity at higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Bamezai
S
S
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Banez
M A
MA
Breslow
E
E
D000433:1-Propanol; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000502:physiology; D011487:Protein Conformation; D011522:Protons; D013329:Structure-Activity Relationship; D014452:Ubiquitins / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology / Q000502:physiology
D011522:Protons; D014452:Ubiquitins; D000433:1-Propanol; D008715:Methionine; D003488:Cyanogen Bromide