DOI: 10.1073/pnas.1201797109. Temperature dependence of protein folding kinetics in living cells

Temperature dependence of protein folding kinetics in living cells

10.1073/pnas.1201797109

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

We measure the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) inside bone tissue cells as a function of temperature from 38 to 48 °C. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min. We find that this method yields improved thermal melts compared to heating a sample chamber or microscope stage. By comparing results for six cells with in vitro data, we show that the protein is stabilized by about 6 kJ/mole in the cytoplasm, but the temperature dependence of folding kinetics is similar to in vitro. The main difference is a slightly steeper temperature dependence of the folding rate in some cells that can be rationalized in terms of temperature-dependent crowding, local viscosity, or hydrophobicity. The observed rate coefficients can be fitted within measurement uncertainty by an effective two-state model, even though PGK folds by a multistate mechanism. We validate the effective two-state model with a three-state free energy landscape of PGK to illustrate that the effective fitting parameters can represent a more complex underlying free energy landscape.

Bibliography

Guo, M., Xu, Y., & Gruebele, M. (2012). Temperature dependence of protein folding kinetics in living cells. Proceedings of the National Academy of Sciences, 109(44), 17863â17867.

Authors 3

Minghao Guo (first)
Yangfan Xu (additional)
Martin Gruebele (additional)

References 31 Referenced 125

10.1126/science.282.5389.740
10.1038/nature01160
10.1126/science.1208351
10.1093/emboj/19.15.3870
10.1016/j.bpj.2008.10.014
10.1073/pnas.1006760107
10.1016/j.bpj.2010.08.066
10.1002/prot.340210302
10.1021/jz101729z
10.1038/nmeth.1435
10.1016/j.bpj.2011.05.071
10.1073/pnas.96.11.6031
10.1074/jbc.M601915200
10.1016/j.jmb.2003.08.011
AR Fersht Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding (W.H. Freeman, New York, 1999). / Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding by Fersht AR (1999)
10.1016/j.jmb.2009.08.016
10.1016/S0968-0004(01)02003-5
10.1002/jps.20417
10.1073/pnas.202331299
10.1073/pnas.0409630102
10.1016/j.bpj.2009.03.066
10.1021/ja200067p
10.1073/pnas.1011354107
10.1073/pnas.0806154105
10.1073/pnas.0709207104
10.1063/1.3262489
10.1016/j.cell.2008.04.030
A Dhar, M Gruebele, Fast relaxation imaging in living cells. Curr Protoc Prot Sci, pp. 28.1.1–28.2.19, Unit 28.1. (2011). / Curr Protoc Prot Sci / Fast relaxation imaging in living cells by Dhar A (2011)
10.1038/nature02046
10.1016/S0065-3233(08)60377-0
10.1063/1.3607605

Dates

Type	When
Created	13 years, 3 months ago (June 5, 2012, 10:05 a.m.)
Deposited	3 years, 4 months ago (April 15, 2022, 10:29 a.m.)
Indexed	3 days ago (Sept. 3, 2025, 5:51 a.m.)
Issued	13 years, 3 months ago (June 4, 2012)
Published	13 years, 3 months ago (June 4, 2012)
Published Online	13 years, 3 months ago (June 4, 2012)
Published Print	12 years, 10 months ago (Oct. 30, 2012)

Funders 0

None

BibTeX

@article{Guo_2012, title={Temperature dependence of protein folding kinetics in living cells}, volume={109}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.1201797109}, DOI={10.1073/pnas.1201797109}, number={44}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Guo, Minghao and Xu, Yangfan and Gruebele, Martin}, year={2012}, month=jun, pages={17863–17867} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        9,
        3
      ]
    ],
    "date-time": "2025-09-03T09:51:00Z",
    "timestamp": 1756893060699
  },
  "reference-count": 31,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "44",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        2012,
        10,
        30
      ]
    ]
  },
  "abstract": "<jats:p>We measure the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) inside bone tissue cells as a function of temperature from 38 to 48\u2009\u00b0C. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min. We find that this method yields improved thermal melts compared to heating a sample chamber or microscope stage. By comparing results for six cells with in vitro data, we show that the protein is stabilized by about 6\u00a0kJ/mole in the cytoplasm, but the temperature dependence of folding kinetics is similar to in vitro. The main difference is a slightly steeper temperature dependence of the folding rate in some cells that can be rationalized in terms of temperature-dependent crowding, local viscosity, or hydrophobicity. The observed rate coefficients can be fitted within measurement uncertainty by an effective two-state model, even though PGK folds by a multistate mechanism. We validate the effective two-state model with a three-state free energy landscape of PGK to illustrate that the effective fitting parameters can represent a more complex underlying free energy landscape.</jats:p>",
  "DOI": "10.1073/pnas.1201797109",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2012,
        6,
        5
      ]
    ],
    "date-time": "2012-06-05T14:05:21Z",
    "timestamp": 1338905121000
  },
  "page": "17863-17867",
  "update-policy": "http://dx.doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 125,
  "title": "Temperature dependence of protein folding kinetics in living cells",
  "prefix": "10.1073",
  "volume": "109",
  "author": [
    {
      "given": "Minghao",
      "family": "Guo",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Departments of Physics,"
        }
      ]
    },
    {
      "given": "Yangfan",
      "family": "Xu",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Chemical and Biomolecular Engineering, and"
        }
      ]
    },
    {
      "given": "Martin",
      "family": "Gruebele",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Departments of Physics,"
        },
        {
          "name": "Chemistry and Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        2012,
        6,
        4
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_3_1_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.282.5389.740"
    },
    {
      "key": "e_1_3_3_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nature01160"
    },
    {
      "key": "e_1_3_3_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1208351"
    },
    {
      "key": "e_1_3_3_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/19.15.3870"
    },
    {
      "key": "e_1_3_3_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.bpj.2008.10.014"
    },
    {
      "key": "e_1_3_3_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.1006760107"
    },
    {
      "key": "e_1_3_3_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.bpj.2010.08.066"
    },
    {
      "key": "e_1_3_3_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/prot.340210302"
    },
    {
      "key": "e_1_3_3_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/jz101729z"
    },
    {
      "key": "e_1_3_3_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nmeth.1435"
    },
    {
      "key": "e_1_3_3_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.bpj.2011.05.071"
    },
    {
      "key": "e_1_3_3_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.96.11.6031"
    },
    {
      "key": "e_1_3_3_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.M601915200"
    },
    {
      "key": "e_1_3_3_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.jmb.2003.08.011"
    },
    {
      "key": "e_1_3_3_15_2",
      "volume-title": "Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding",
      "author": "Fersht AR",
      "year": "1999",
      "unstructured": "AR Fersht Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding (W.H. Freeman, New York, 1999)."
    },
    {
      "key": "e_1_3_3_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.jmb.2009.08.016"
    },
    {
      "key": "e_1_3_3_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0968-0004(01)02003-5"
    },
    {
      "key": "e_1_3_3_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/jps.20417"
    },
    {
      "key": "e_1_3_3_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.202331299"
    },
    {
      "key": "e_1_3_3_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.0409630102"
    },
    {
      "key": "e_1_3_3_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.bpj.2009.03.066"
    },
    {
      "key": "e_1_3_3_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ja200067p"
    },
    {
      "key": "e_1_3_3_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.1011354107"
    },
    {
      "key": "e_1_3_3_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.0806154105"
    },
    {
      "key": "e_1_3_3_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.0709207104"
    },
    {
      "key": "e_1_3_3_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1063/1.3262489"
    },
    {
      "key": "e_1_3_3_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.cell.2008.04.030"
    },
    {
      "key": "e_1_3_3_28_2",
      "first-page": "28.1.1",
      "article-title": "Fast relaxation imaging in living cells",
      "author": "Dhar A",
      "year": "2011",
      "unstructured": "A Dhar, M Gruebele, Fast relaxation imaging in living cells. Curr Protoc Prot Sci, pp. 28.1.1\u201328.2.19, Unit 28.1. (2011).",
      "journal-title": "Curr Protoc Prot Sci"
    },
    {
      "key": "e_1_3_3_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nature02046"
    },
    {
      "key": "e_1_3_3_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0065-3233(08)60377-0"
    },
    {
      "key": "e_1_3_3_31_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1063/1.3607605"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.1201797109",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2022,
        4,
        15
      ]
    ],
    "date-time": "2022-04-15T14:29:11Z",
    "timestamp": 1650032951000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/full/10.1073/pnas.1201797109"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2012,
        6,
        4
      ]
    ]
  },
  "references-count": 31,
  "journal-issue": {
    "issue": "44",
    "published-print": {
      "date-parts": [
        [
          2012,
          10,
          30
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.1201797109"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.1201797109",
  "relation": {
    "has-review": [
      {
        "id-type": "doi",
        "id": "10.3410/f.717847807.793153366",
        "asserted-by": "object"
      }
    ]
  },
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        2012,
        6,
        4
      ]
    ]
  },
  "assertion": [
    {
      "value": "2012-06-04",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}