The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.
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)
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