The histidine‐phosphocarrier protein ofStreptomyces coelicolorfolds by a partially folded species at low pH

Abstract

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°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 DG ¼ 4.0 ± 0.2 kcalAEmol )1 (1 kcal ¼ 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 pK a ¼ 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.

Residues His-15 and Arg-17 of HPr Participate Differently in Catabolite Signal Processing via CcpA

“…3). The spectra are identical with each other and the ones of E. coli and Streptomyces coelicolor HPr (23,24). We therefore assume that all proteins are correctly folded.…”

The Helical Structure Propensity in the First Helix of the Histidine Phosphocarrier Protein of Streptomyces coelicolor

“…The M1L mutant was expressed and purified as the wildtype protein, with similar yields [8]. The His 6 -tag was removed with thrombin.…”

“…The presence of the different components of the PTS in S. coelicolor has been reported, and the corresponding proteins cloned and expressed [6,7]. We have undertaken an extensive description of the structures and conformational stabilities of the HPr and EI proteins in S. coelicolor [8][9][10][11]. HPr contains 93 amino acid residues; it lacks cysteine and tyrosine residues, and it only contains one tryptophan and one phenylalanine residues, close to the active site histidine.…”

Structure and conformational stability of the enzyme I of Streptomyces coelicolor explored by FTIR and circular dichroism

“…All spectra were corrected by subtracting the proper baseline. The mean residue ellipticity, [H], was obtained from the raw ellipticity data, H, as reported elsewhere [10]. The helical content of scEI was calculated from its mean residue ellipticity at 222 nm [15]:…”

“…There is a growing interest in determining to which extent related proteins of the same family share the same conformational stability features [9]. For instance, we have shown that HPr of S. coelicolor shows different stability and folding properties than those HPrs of B. subtilis and E. coli [10,11], although the structures are similar. Stability studies of EI from E. coli [4,12] and M. capricolum [3,13] have been carried out using several spectroscopic techniques, namely, fluorescence and CD, and calorimetry (DSC).…”