Guanidine hydrochloride induced equilibrium unfolding studies of colicin B and its channel-forming fragment.
The conformational stabilities of full-length colicin B and its isolated C-terminal domain were studied by guanidine hydrochloride induced unfolding. The unfolding/refolding was monitored by far-UV CD and intrinsic tryptophan fluorescence spectroscopies. At pH 7.4, the disruption of the secondary structure of full-length colicin B is monophasic, while changes in tertiary structure occur in two separate transitions. The intermediate species, which is well-populated around 2.2 M guanidine hydrochloride, exhibits secondary and tertiary structures distinct from both native and unfolded states. Whereas the domain structure of native full-length colicin B is reflected in its DSC profile, the folding intermediate of the same protein exhibits a single unresolved peak. These observations have led us to propose an unfolding model for full-length colicin B where the first transition between 0 and 2.5 M GuHCl with an associated free energy of 3 kcal/mol correlates with the partial unfolding of the R/T domain. The stability of full-length colicin B is weakened due to the presence of the R/T domain in both the native [Ortega, A., Lambotte, S., and Bechinger, B. (2001) J. Biol. Chem. 276 (17), 13563-13572] and the intermediate states. The second transition between 2.5 and 5 M GuHCl involves unfolding of the C-terminal domain (Delta = 7 kcal/mol). The isolated colicin B C-terminal domain consists of two subdomains, and the two parts of this protein fragment unfold sequentially through the formation of at least one intermediate. The significance of these results for membrane insertion of colicin B is discussed.
Sathish
H A
HA
Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany.
Cusan
Monica
M
Aisenbrey
Christopher
C
Bechinger
Burkhard
B
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D003087:Colicins; D010446:Peptide Fragments; D014364:Tryptophan; D019791:Guanidine