Characterization of a transient unfolding intermediate in a core mutant of γs-Crystallin

Bryon Mahler, Kiran Doddapaneni, Ian Kleckner, Chunhua Yuan, Graeme Wistow, Zhengrong Wu

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T 2-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.

Original languageEnglish (US)
Pages (from-to)840-850
Number of pages11
JournalJournal of Molecular Biology
Volume405
Issue number3
DOIs
StatePublished - Jan 21 2011
Externally publishedYes

Fingerprint

Crystallins
Proteins
Temperature
Deuterium
Fluorescence Spectrometry
Mutant Proteins
Cataract
Lenses
Urea
Hydrogen
Magnetic Resonance Spectroscopy
Light
Mutation
Population

Keywords

  • γS-crystallin
  • cataract
  • protein unfolding
  • relaxation dispersion

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Characterization of a transient unfolding intermediate in a core mutant of γs-Crystallin. / Mahler, Bryon; Doddapaneni, Kiran; Kleckner, Ian; Yuan, Chunhua; Wistow, Graeme; Wu, Zhengrong.

In: Journal of Molecular Biology, Vol. 405, No. 3, 21.01.2011, p. 840-850.

Research output: Contribution to journalArticle

Mahler, Bryon ; Doddapaneni, Kiran ; Kleckner, Ian ; Yuan, Chunhua ; Wistow, Graeme ; Wu, Zhengrong. / Characterization of a transient unfolding intermediate in a core mutant of γs-Crystallin. In: Journal of Molecular Biology. 2011 ; Vol. 405, No. 3. pp. 840-850.
@article{d49878f445f24ef08e5b90339342cee2,
title = "Characterization of a transient unfolding intermediate in a core mutant of γs-Crystallin",
abstract = "In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T 2-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2{\%}, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.",
keywords = "γS-crystallin, cataract, protein unfolding, relaxation dispersion",
author = "Bryon Mahler and Kiran Doddapaneni and Ian Kleckner and Chunhua Yuan and Graeme Wistow and Zhengrong Wu",
year = "2011",
month = "1",
day = "21",
doi = "10.1016/j.jmb.2010.11.005",
language = "English (US)",
volume = "405",
pages = "840--850",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Characterization of a transient unfolding intermediate in a core mutant of γs-Crystallin

AU - Mahler, Bryon

AU - Doddapaneni, Kiran

AU - Kleckner, Ian

AU - Yuan, Chunhua

AU - Wistow, Graeme

AU - Wu, Zhengrong

PY - 2011/1/21

Y1 - 2011/1/21

N2 - In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T 2-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.

AB - In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T 2-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.

KW - γS-crystallin

KW - cataract

KW - protein unfolding

KW - relaxation dispersion

UR - http://www.scopus.com/inward/record.url?scp=78650871880&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78650871880&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2010.11.005

DO - 10.1016/j.jmb.2010.11.005

M3 - Article

C2 - 21108948

AN - SCOPUS:78650871880

VL - 405

SP - 840

EP - 850

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -