synthetic biology of proteins tuning gfps folding and stability with fluoroproline合成生物学与fluoroproline调优gfp的蛋白质的折叠和稳定性.pdfVIP

Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline Thomas Steiner, Petra Hess¤a, Jae Hyun Bae¤b, Birgit Wiltschi, Luis Moroder, Nediljko Budisa* Max Planck Institute of Biochemistry, Martinsried, Germany Abstract c Background: Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C - exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. Cc-fluorination. Synthetic chemistry has routinely exploited ring- substituted proline analogs in order to change, modulate or control folding and stability of peptides. Methodology/Principal Findings: In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the si