The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pH.
The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 degrees C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is deltaG = 4.0 +/- 0.2 kcal x mol-1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7-9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5-4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 +/- 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family.
Fernández-Ballester
Gregorio
G
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche (Alicante), Spain.
Maya
Javier
J
Martín
Alejandro
A
Parche
Stephan
S
Gómez
Javier
J
Titgemeyer
Fritz
F
Neira
José L
JL
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D001426:Bacterial Proteins; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013302:Streptomyces / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; C027132:1-anilino-8-naphthalenesulfonate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr