Equilibrium phi-analysis of a molten globule: the 1-149 apoflavodoxin fragment.
The apoflavodoxin fragment comprising residues 1-149 that can be obtained by chemical cleavage of the C-terminal alpha-helix of the full-length protein is known to populate a molten globule conformation that displays a cooperative behaviour and experiences two-state urea and thermal denaturation. Here, we have used a recombinant form of this fragment to investigate molten globule energetics and to derive structural information by equilibrium Phi-analysis. We have characterized 15 mutant fragments designed to probe the persistence of native interactions in the molten globule and compared their conformational stability to that of the equivalent full-length apoflavodoxin mutants. According to our data, most of the mutations analysed modify the stability of the molten globule fragment following the trend observed when the same mutations are implemented in the full-length protein. However, the changes in stability observed in the molten globule are much smaller and the Phi-values calculated are (with a single exception) below 0.4. This is consistent with an overall and significant debilitation of the native structure. Nevertheless, the fact that the molten globule fragment can be stabilised using as a guide the native structure of the full-length protein (by increasing helix propensity, optimising charge interactions and filling small cavities) suggests that the overall structure of the molten globule is still quite close to native, in spite of the lowered stability observed.
López-Llano
J
J
Dep. Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and Biocomputation and Complex Systems Physics Institute-BIFI, University of Zaragoza, Spain.
Campos
L A
LA
Bueno
M
M
Sancho
J
J
D017033:Anabaena / Q000737:chemistry; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D001059:Apoproteins; D001426:Bacterial Proteins; D005418:Flavodoxin; D010446:Peptide Fragments; C026932:apoflavodoxin