Modulation of Intracellular Protein Activity at Level of Protein Folding by Beta-turn Engineering

다국어 초록 (Multilingual Abstract)

Control of the intracellular protein activity is veryimportant in various biological studies and biotechnology.
This has generally been achieved at the transcription andtranslation levels. Although control of the intracellularactivity at the protein folding level is conceptually possible,but there have been few studies. The present studyexamined this possibility by modulating the in vivo proteinfolding rate of green fluorescence protein (GFP) throughbeta-turn engineering. A type II’ two residue beta-turn inGFP was targeted to generate two sets of mutants. First, aswitch-off mutant was designed to stop the protein activitycompletely. The modulation mutants were then constructedto change the rates of GFP folding. The design of mutantswas based on the rationale that residues i+1 and i+2 of abeta-turn have defined residue preferences, and theirperturbation affects the rate of protein folding. The in vivofluorescence activity of the designed GFP variants wasswitched off and modulated as expected. The change in thein vivo folding patterns of the mutants was confirmed bySDS-PAGE and found to be similar to the intracellularfluorescence activities of the mutants. The in vitro refoldingkinetics performed with purified variants showed correlationswith the in vivo folding patterns. These results showed thatthe beta-turns in a protein can be a target for modulatingthe in vivo protein folding pattern and activity.

참고문헌 (Reference)

1 Ecroyd, H., "Unraveling the mysteries of protein folding and misfolding" 60 : 769-774, 2008

2 Arpino, J. A., "Tuning the dials of synthetic biology" 159 : 1236-1253, 2013

3 Lane, T. J., "To milliseconds and beyond: Challenges in the simulation of protein folding" 23 : 58-65, 2013

4 de Alba, E., "The turn sequence directs beta-strand alignment in designed beta-hairpins" 8 : 2234-2244, 1999

5 Yang, F., "The molecular structure of green fluorescent protein" 14 : 1246-1251, 1996

6 Madan, B., "Structural and sequence features of two residue turns in beta-hairpins" 2014

7 Grünberg, R., "Strategies for protein synthetic biology" 38 : 2663-2675, 2010

8 Venkatachalam, C. M., "Stereochemical criteria for polypeptides and proteins. V. Conformation of a system of three linked peptide units" 6 : 1425-1436, 1968

9 Marcelino, A. M., "Roles of beta-turns in protein folding: From peptide models to protein engineering" 89 : 380-391, 2008

10 Ramirez-Alvarado, M., "Role of beta-turn residues in beta-hairpin formation and stability in designed peptides" 273 : 898-912, 1997

11 Sugita, Y., "Replica-exchange molecular dynamics method for protein folding" 314 : 141-151, 1999

12 Huang, J., "Relationship between protein folding kinetics and amino acid properties" 43 : 567-572, 2012

13 Blount, B. A., "Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology" 7 : e33279-, 2012

14 Cabrita, L. D., "Protein folding on the ribosome" 20 : 33-45, 2010

15 Palaniyandi Muthukutty, "Production of Human Papillomavirus 6b L1 Virus-like Particles Incorporated with Enhanced Green Fluorescent Whole Protein in Silkworm Larvae" 한국생물공학회 18 (18): 514-519, 2013

16 Anfinsen, C. B., "Principles that govern the folding of protein chains" 181 : 223-230, 1973

17 Kaufmann, K. W., "Practically useful: What the rosetta protein modeling suite can do for you" 49 : 2987-2998, 2010

18 Olson, E. J., "Post-translational tools expand the scope of synthetic biology" 16 : 300-306, 2012

19 Martinez, J. C., "Obligatory steps in protein folding and the conformational diversity of the transition state" 5 : 721-729, 1998

20 Raghunathan, G., "Modulation of protein stability and aggregation properties by surface charge engineering" 9 : 2379-2389, 2013

21 Merkel, J. S., "Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel β strands of green fluorescent protein" 275 : 29200-29206, 2000

22 Matouschek, A., "Mapping the transition state and pathway of protein folding by protein engineering" 340 : 122-126, 1989

23 Sriram Sokalingam, "In silico Study on the Effect of Surface Lysines and Arginines on the Electrostatic Interactions and Protein Stability" 한국생물공학회 18 (18): 18-26, 2013

24 Snow, C. D., "How well can simulation predict protein folding kinetics and thermodynamics?" 34 : 43-69, 2005

25 Anne Tondervik, "High Production of Recombinant Norwegian Salmonid Alphavirus E1 and E2 Proteins in Escherichia coli by Fusion to Secretion Signal Sequences and Removal of Hydrophobic Domains" 한국생물공학회 18 (18): 742-750, 2013

26 Pronk, S., "GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit" 29 : 845-854, 2013

27 Bussi, G., "Free-energy landscape for β hairpin folding from combined parallel tempering and metadynamics" 128 : 13435-13441, 2006

28 Cormack, B. P., "FACS-optimized mutants of the green fluorescent protein (GFP)" 173 : 33-38, 1996

29 Simpson, E. R., "Engineering diverse changes in beta-turn propensities in the N-terminal beta-hairpin of ubiquitin reveals significant effects on stability and kinetics but a robust folding transition state" 45 : 4220-4230, 2006

30 McCallister, E. L., "Critical role of beta-hairpin formation in protein G folding" 7 : 669-673, 2000

31 Hartl, F. U., "Converging concepts of protein folding in vitro and in vivo" 16 : 574-581, 2009

32 Spiwok, V., "Continuous metadynamics in essential coordinates as a tool for free energy modelling of conformational changes" 14 : 995-1002, 2008

33 Chou, P. Y., "Conservation of chain reversal regions in proteins" 26 : 385-399, 1979

34 Sibanda, B. L., "Conformation of β-hairpins in protein structures: A systematic classification with applications to modelling by homology, electron density fitting and protein engineering" 206 : 759-777, 1989

35 Lewis, P. N., "Chain reversals in proteins" 303 : 211-229, 1973

36 Gunasekaran, K., "Beta-hairpins in proteins revisited: Lessons for de novo design" 10 : 1131-1141, 1997

37 Sokalingam, S., "A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluo-rescent protein" 7 : e40410-, 2012

38 Hutchinson, E. G., "A revised set of potentials for beta-turn formation in proteins" 3 : 2207-2216, 1994

39 Govindan Raghunathan, "A Comparative Study on the Stability and Structure of Two Different Green Fluorescent Proteins in Organic Co-solvent Systems" 한국생물공학회 18 (18): 342-349, 2013