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PreprintCited by 1Protein Structure and DynamicsHeat shock proteins researchBiochemical and Structural Characterization

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Design of stable circular permutants of the GroEL chaperone apical domain

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Melnik, Tatiana N., Majorina, Maria A., Vorobeva, Daria E., Nagibina, Galina S., Veselova, Victoria R., Glukhova, Ksenia А., Pak, Marina A., Ivankov, Dmitry N., Uversky, Vladimir N., Melnik, Bogdan S.·2023·Cell Communication and Signaling
DOI 10.21203/rs.3.rs-3360046/v1PMC PMC10836027

Preprint — published as 10.1186/s12964-023-01426-4 · Paper page

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Abstract

Enhancing protein stability holds paramount significance in biotechnology, therapeutics, and the food industry. Circular permutations offer a distinctive avenue for manipulating protein stability while keeping intra-protein interactions intact. Amidst the creation of circular permutants, determining the optimal placement of the new N- and C-termini stands as a pivotal, albeit largely unexplored, endeavor. In this study, we employed PONDR-FIT’s predictions of disorder propensity to guide the design of circular permutants for the GroEL apical domain (residues 191–345). Our underlying hypothesis posited that a higher predicted disorder value would correspond to reduced stability in the circular permutants, owing to the increased likelihood of fluctuations in the novel N- and C-termini. To substantiate this hypothesis, we engineered six circular permutants, positioning glycines within the loops as locations for the new N- and C-termini. We demonstrated the validity of our hypothesis along the set of the designed circular permutants, as supported by measurements of melting temperatures by circular dichroism and differential scanning microcalorimetry. Consequently, we propose a novel computational methodology that rationalizes the design of circular permutants with projected stability.

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Research works printed but not linked: 49

  1. Khersonsky O, Lipsh R, Avizemer Z, Ashani Y, Goldsmith M, Leader H et al. Automated Design of Efficient and Functionally Diverse Enzyme Repertoires. Mol Cell. 2018;72(1):178 – 86 e5. 10.1016/j.molcel.2018.08.033. · DOI
  2. Rothlisberger D, Khersonsky O, Wollacott AM, Jiang L, DeChancie J, Betker J, et al. Kemp elimination catalysts by computational enzyme design. Nature. 2008;453(7192):190–5. 10.1038/nature06879. · DOI
  3. Khoury GA, Smadbeck J, Kieslich CA, Floudas CA. Protein folding and de novo protein design for biotechnological applications. Trends Biotechnol. 2014;32(2):99–109. 10.1016/j.tibtech.2013.10.008. · DOI
  4. Chevalier A, Silva DA, Rocklin GJ, Hicks DR, Vergara R, Murapa P, et al. Massively parallel de novo protein design for targeted therapeutics. Nature. 2017;550(7674):74–9. 10.1038/nature23912. · DOI · Paper page
  5. Tang N, Liu J, Cheng Y. Potential improvement of the thermal stability of sweet-tasting proteins by structural calculations. Food Chem. 2021;345:128750. 10.1016/j.foodchem.2020.128750. · DOI
  6. Wang R, Wang S, Xu Y, Yu X. Enhancing the thermostability of Rhizopus chinensis lipase by rational design and MD simulations. Int J Biol Macromol. 2020;160:1189–200. 10.1016/j.ijbiomac.2020.05.243. · DOI
  7. Clifton BE, Whitfield JH, Sanchez-Romero I, Herde MK, Henneberger C, Janovjak H, et al. Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors. Methods Mol Biol. 2017;1596:71–87. 10.1007/978-1-4939-6940-1_5. · DOI
  8. Reitinger S, Yu Y, Wicki J, Ludwiczek M, D'Angelo I, Baturin S, et al. Circular permutation of Bacillus circulans xylanase: a kinetic and structural study. Biochemistry. 2010;49(11):2464–74. 10.1021/bi100036f. · DOI
  9. Ferreira GC, Cheltsov AV. Circular permutation of 5-aminolevulinate synthase as a tool to evaluate folding, structure and function. Cell Mol Biol (Noisy-le-grand). 2002;48(1):11–6.
  10. Heinemann U, Hahn M. Circular permutation of polypeptide chains: implications for protein folding and stability. Prog Biophys Mol Biol. 1995;64(2–3):121–43. 10.1016/0079-6107(95)00013-5. · DOI
  11. Abdullaev ZK, Latypov RF, Badretdinov AY, Dolgikh DA, Finkelstein AV, Uversky VN, et al. S6 permutein shows that the unusual target topology is not responsible for the absence of rigid tertiary structure in de novo protein albebetin. FEBS Lett. 1997;414(2):243–6. 10.1016/s0014-5793(97)01042-9. · DOI
  12. Uversky VN, Kutyshenko VP, Protasova N, Rogov VV, Vassilenko KS, Gudkov AT. Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands. Protein Sci. 1996;5(9):1844–51. 10.1002/pro.5560050910. · DOI
  13. Protasova N, Kireeva ML, Murzina NV, Murzin AG, Uversky VN, Gryaznova OI, et al. Circularly permuted dihydrofolate reductase of E. coli has functional activity and a destabilized tertiary structure. Protein Eng. 1994;7(11):1373–7. 10.1093/protein/7.11.1373. · DOI
  14. Lo WC, Dai T, Liu YY, Wang LF, Hwang JK, Lyu PC. Deciphering the preference and predicting the viability of circular permutations in proteins. PLoS ONE. 2012;7(2):e31791. 10.1371/journal.pone.0031791. · DOI
  15. Butler JS, Mitrea DM, Mitrousis G, Cingolani G, Loh SN. Structural and thermodynamic analysis of a conformationally strained circular permutant of barnase. Biochemistry. 2009;48(15):3497–507. 10.1021/bi900039e. · DOI
  16. Iwakura M, Nakamura T. Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase. Protein Eng. 1998;11(8):707–13. 10.1093/protein/11.8.707. · DOI
  17. Paszkiewicz KH, Sternberg MJ, Lappe M. Prediction of viable circular permutants using a graph theoretic approach. Bioinformatics. 2006;22(11):1353–8. 10.1093/bioinformatics/btl095. · DOI
  18. Xue B, Dunbrack RL, Williams RW, Dunker AK, Uversky VN. PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim Biophys Acta. 2010;1804(4):996–1010. 10.1016/j.bbapap.2010.01.011. · DOI
  19. Ishida T, Kinoshita K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res. 2007;W460–4. 10.1093/nar/gkm363. 35(Web Server issue). · DOI
  20. Melnik BS, Povarnitsyna TV, Glukhov AS, Melnik TN, Uversky VN, Sarma RH. SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP. J Biomol Struct Dyn. 2012;29(4):815–24. 10.1080/07391102.2012.10507414. · DOI
  21. Nagibina GS, Tin UF, Glukhov AS, Melnik TN, Melnik BS. Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridge in Galphao Protein. Protein Pept Lett. 2016;23(2):176–84. 10.2174/092986652302160105130540. · DOI
  22. Nagibina GS, Melnik TN, Glukhova KA, Uversky VN, Melnik BS. Intrinsic disorder-based design of stable globular proteins. Prog Mol Biol Transl Sci. 2020;174:157–86. 10.1016/bs.pmbts.2020.05.005. · DOI
  23. Nagibina GS, Marchenkov VV, Glukhova KA, Melnik TN, Melnik BS. Verification of the Stabilized Protein Design Based on the Prediction of Intrinsically Disordered Regions: Ribosomal Proteins L1. Biochem (Mosc). 2020;85(1):90–8. 10.1134/S0006297920010083. · DOI
  24. Nagibina GS, Glukhova KA, Uversky VN, Melnik TN, Melnik BS. Intrinsic Disorder-Based Design of Stable Globular Proteins. Biomolecules. 2019;10(1). 10.3390/biom10010064. · DOI
  25. Braig K, Otwinowski Z, Hegde R, Boisvert DC, Joachimiak A, Horwich AL, et al. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature. 1994;371(6498):578–86. 10.1038/371578a0. · DOI
  26. Feltham JL, Gierasch LM. GroEL-substrate interactions: molding the fold, or folding the mold? Cell. 2000;100(2):193–6. 10.1016/s0092-8674(00)81557-3. · DOI
  27. Chen L, Sigler PB. The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity. Cell. 1999;99(7):757–68. 10.1016/s0092-8674(00)81673-6. · DOI
  28. Tanaka N, Fersht AR. Identification of substrate binding site of GroEL minichaperone in solution. J Mol Biol. 1999;292(1):173–80. 10.1006/jmbi.1999.3041. · DOI
  29. Sigler PB, Xu Z, Rye HS, Burston SG, Fenton WA, Horwich AL. Structure and function in GroEL-mediated protein folding. Annu Rev Biochem. 1998;67:581–608. 10.1146/annurev.biochem.67.1.581. · DOI
  30. Xu Z, Horwich AL, Sigler PB. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature. 1997;388(6644):741–50. 10.1038/41944. · DOI
  31. Llorca O, Marco S, Carrascosa JL, Valpuesta JM. Symmetric GroEL-GroES complexes can contain substrate simultaneously in both GroEL rings. FEBS Lett. 1997;405(2):195–9. 10.1016/s0014-5793(97)00186-5. · DOI
  32. Smoot AL, Panda M, Brazil BT, Buckle AM, Fersht AR, Horowitz PM. The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL. Biochemistry. 2001;40(14):4484–92. 10.1021/bi001822b. · DOI
  33. Wang Q, Buckle AM, Foster NW, Johnson CM, Fersht AR. Design of highly stable functional GroEL minichaperones. Protein Sci. 1999;8(10):2186–93. 10.1110/ps.8.10.2186. · DOI
  34. Chatellier J, Hill F, Lund PA, Fersht AR. In vivo activities of GroEL minichaperones. Proc Natl Acad Sci U S A. 1998;95(17):9861–6. 10.1073/pnas.95.17.9861. · DOI
  35. Golbik R, Zahn R, Harding SE, Fersht AR. Thermodynamic stability and folding of GroEL minichaperones. J Mol Biol. 1998;276(2):505–15. 10.1006/jmbi.1997.1538. · DOI
  36. Marchenkov V, Gorokhovatsky A, Marchenko N, Ivashina T, Semisotnov G. Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution. Biomolecules. 2020;10(1). 10.3390/biom10010162. · DOI
  37. Kamireddi M, Eisenstein E, Reddy P. Stable expression and rapid purification of Escherichia coli GroEL and GroES chaperonins. Protein Expr Purif. 1997;11(1):47–52. 10.1006/prep.1997.0764. · DOI
  38. Wolf P. A critical reappraisal of Waddell's technique for ultraviolet spectrophotometric protein estimation. Anal Biochem. 1983;129(1):145–55. 10.1016/0003-2697(83)90062-3. · DOI
  39. Senin A, Potekhin S, Tiktopulo E, Filomonov V. Differential scanning microcalorimeter SCAL-1. J Therm Anal Calorim. 2000;62:153–60. · DOI
  40. Privalov PL, Potekhin SA. Scanning microcalorimetry in studying temperature-induced changes in proteins. Methods Enzymol. 1986;131:4–51. 10.1016/0076-6879(86)31033-4. · DOI
  41. Chatellier J, Hill F, Foster NW, Goloubinoff P, Fersht AR. From minichaperone to GroEL 3: properties of an active single-ring mutant of GroEL. J Mol Biol. 2000;304(5):897–910. 10.1006/jmbi.2000.4278. · DOI
  42. Chatellier J, Hill F, Fersht AR. From minichaperone to GroEL 2: importance of avidity of the multisite ring structure. J Mol Biol. 2000;304(5):883–96. 10.1006/jmbi.2000.4277. · DOI
  43. Zhang Y, Skolnick J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 2005;33(7):2302–9. 10.1093/nar/gki524. · DOI · Paper page
  44. Pak MA, Markhieva KA, Novikova MS, Petrov DS, Vorobyev IS, Maksimova ES, et al. Using AlphaFold to predict the impact of single mutations on protein stability and function. PLoS ONE. 2023;18(3):e0282689. 10.1371/journal.pone.0282689. · DOI · Paper page
  45. Pak MA, Ivankov DN. Best templates outperform homology models in predicting the impact of mutations on protein stability. Bioinformatics. 2022;38(18):4312–20. 10.1093/bioinformatics/btac515. · DOI · Paper page
  46. Tsaban T, Varga JK, Avraham O, Ben-Aharon Z, Khramushin A, Schueler-Furman O. Harnessing protein folding neural networks for peptide-protein docking. Nat Commun. 2022;13(1):176. 10.1038/s41467-021-27838-9. · DOI · Paper page
  47. Buel GR, Walters KJ. Can AlphaFold2 predict the impact of missense mutations on structure? Nat Struct Mol Biol. 2022;29(1):1–2. 10.1038/s41594-021-00714-2. · DOI · Paper page
  48. Outeiral C, Nissley DA, Deane CM. Current structure predictors are not learning the physics of protein folding. Bioinformatics. 2022;38(7):1881–7. 10.1093/bioinformatics/btab881. · DOI · Paper page
  49. Finkelstein AV. Does AlphaFold predict the spatial structure of a protein from physics or recognize it (its main parts and their association) using databases? bioRxiv. 2022:2022.11. 21.517308.

Metadata

Authors
Melnik, T. N., Majorina, M. A., Vorobeva, D. E., Nagibina, G. S., Veselova, V. R., Glukhova, K. А., Pak, M. A., Ivankov, D. N., Uversky, V. N., & Melnik, B. S.
Affiliations
  • Russian Academy of Sciences (RU)
  • Skolkovo Institute of Science and Technology (RU)
  • Moscow Institute of Physics and Technology (RU)
Corresponding author
  • Uversky, Vladimir N.
  • Melnik, Bogdan S.
ORCID
Venue
Cell Communication and Signaling
Publisher
Research Square Platform LLC
Published
2023-10-09
Volume
22
Issue
1
Pages
90-90
ISSN
1478-811X (ISSN-L)
DOI
10.21203/rs.3.rs-3360046/v1
PMC
PMC10836027
OpenAlex
W4391450021
Cited by
1
References
1
Citations by year
2025: 1
FWCI
0.14
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Communication and Signaling (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
Russian Science Foundation (21–14–00268)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
circular permutation, GroEL, protein stability, intrinsically disordered regions, melting point, protein engineering

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