Folding behavior of a backbone-reversed protein: reversible polyproline type II to beta-sheet thermal transitions in retro-GroES multimers with GroES-like features.
The structural consequences of the reversal of polypeptide backbone direction (retro modification) remain insufficiently explored. Here, we describe the behavior of an engineered, backbone-reversed form of the 97 residues-long GroES co-chaperonin of Escherichia coli. FTIR and far-UV CD spectroscopy suggest that retro-GroES adopts a mixed polyproline type II (PPII)-beta-strand structure with a beta(II) type CD spectrum similar to that of GroES. Gel-filtration chromatography reveals that the protein adopts trimeric and/or pentameric quaternary structures, with solubility retained up to concentrations of 5.0-5.5 mg/ml in aqueous solutions. Mutations inserting a single tryptophan residue as a spectroscopic probe at three different sites cause no perturbation in the protein's CD spectral characteristics, or in its quaternary structural status. The protein is cooperatively dissociated, and non-cooperatively unfolded, by both guanidine hydrochloride and urea. Intriguingly, unlike with GroES, retro-GroES is not unfolded by heat. Instead, there is a reversible structural transition involving conversion of PPII structure to beta sheet structure, upon heating, with no attendant aggregation even at 90 degrees C. Retro-GroES does not bind GroEL. In summary, some structure-forming characteristics of GroES appear to be conserved through the backbone reversal process, although the differential conformational behavior upon heating also indicates differences.
Ahmed
Shubbir
S
Protein Science and Engineering Division, Institute of Microbial Technology, Chandigarh 160036, India.
Shukla
Anshuman
A
Guptasarma
Purnananda
P
D018835:Chaperonin 10 / Q000737:chemistry*; D018834:Chaperonin 60 / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013816:Thermodynamics
D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D010455:Peptides; C011083:polyproline