Investigating the effects of mutations on protein aggregation in the cell - PubMed

Affiliations

• PMID: 15611128
• DOI: 10.1074/jbc.M412951200

Free article

Investigating the effects of mutations on protein aggregation in the cell

Giulia Calloni et al. J Biol Chem. 2005.

Free article

Abstract

The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.

Similar articles

• Low-level expression of a folding-incompetent protein in Escherichia coli: search for the molecular determinants of protein aggregation in vivo.

  Winkelmann J, Calloni G, Campioni S, Mannini B, Taddei N, Chiti F. Winkelmann J, et al. J Mol Biol. 2010 May 14;398(4):600-13. doi: 10.1016/j.jmb.2010.03.030. Epub 2010 Mar 25. J Mol Biol. 2010. PMID: 20346957

• The induction of α-helical structure in partially unfolded HypF-N does not affect its aggregation propensity.

  Ahmad B, Vigliotta I, Tatini F, Campioni S, Mannini B, Winkelmann J, Tiribilli B, Chiti F. Ahmad B, et al. Protein Eng Des Sel. 2011 Jul;24(7):553-63. doi: 10.1093/protein/gzr018. Epub 2011 Apr 25. Protein Eng Des Sel. 2011. PMID: 21518735

• Kinetic partitioning of protein folding and aggregation.

  Chiti F, Taddei N, Baroni F, Capanni C, Stefani M, Ramponi G, Dobson CM. Chiti F, et al. Nat Struct Biol. 2002 Feb;9(2):137-43. doi: 10.1038/nsb752. Nat Struct Biol. 2002. PMID: 11799398

• Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology.

  Schlieker C, Bukau B, Mogk A. Schlieker C, et al. J Biotechnol. 2002 Jun 13;96(1):13-21. doi: 10.1016/s0168-1656(02)00033-0. J Biotechnol. 2002. PMID: 12142139 Review.

• Therapeutic protein aggregation: mechanisms, design, and control.

  Roberts CJ. Roberts CJ. Trends Biotechnol. 2014 Jul;32(7):372-80. doi: 10.1016/j.tibtech.2014.05.005. Epub 2014 Jun 4. Trends Biotechnol. 2014. PMID: 24908382 Free PMC article. Review.

Cited by

• The fast-folding HP35 double mutant has a substantially reduced primary folding free energy barrier.

  Lei H, Deng X, Wang Z, Duan Y. Lei H, et al. J Chem Phys. 2008 Oct 21;129(15):155104. doi: 10.1063/1.2995987. J Chem Phys. 2008. PMID: 19045234 Free PMC article.

• SDM--a server for predicting effects of mutations on protein stability and malfunction.

  Worth CL, Preissner R, Blundell TL. Worth CL, et al. Nucleic Acids Res. 2011 Jul;39(Web Server issue):W215-22. doi: 10.1093/nar/gkr363. Epub 2011 May 18. Nucleic Acids Res. 2011. PMID: 21593128 Free PMC article.

• Sequence determinants of protein aggregation: tools to increase protein solubility.

  Ventura S. Ventura S. Microb Cell Fact. 2005 Apr 22;4(1):11. doi: 10.1186/1475-2859-4-11. Microb Cell Fact. 2005. PMID: 15847694 Free PMC article.

• RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution.

  Kopsidas G, Carman RK, Stutt EL, Raicevic A, Roberts AS, Siomos MA, Dobric N, Pontes-Braz L, Coia G. Kopsidas G, et al. BMC Biotechnol. 2007 Apr 11;7:18. doi: 10.1186/1472-6750-7-18. BMC Biotechnol. 2007. PMID: 17425805 Free PMC article.

• Accurate prediction of functional, structural, and stability changes in PITX2 mutations using in silico bioinformatics algorithms.

  Seifi M, Walter MA. Seifi M, et al. PLoS One. 2018 Apr 17;13(4):e0195971. doi: 10.1371/journal.pone.0195971. eCollection 2018. PLoS One. 2018. PMID: 29664915 Free PMC article.

Publication types

MeSH terms

Substances

LinkOut - more resources

• Full Text Sources

  • Elsevier Science

• Other Literature Sources

  • The Lens - Patent Citations Database