Deamidation, but not truncation, decreases the urea stability of a lens structural protein, βB1-crystallin

Yung Hae Kim, Deborah M. Kapfer, Jos Boekhorst, Nicolette H. Lubsen, Hans Peter Bächinger, Thomas (Tom) Shearer, Larry David, Jimmy B. Feix, Kirsten Lampi

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type βb1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (βb1(ΔN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens βB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.

Original languageEnglish (US)
Pages (from-to)14076-14084
Number of pages9
JournalBiochemistry
Volume41
Issue number47
DOIs
StatePublished - Nov 26 2002

Fingerprint

Crystallins
Denaturation
Urea
Lenses
Cataract
Proteins
Calpain
Electron Spin Resonance Spectroscopy
Post Translational Protein Processing
Circular Dichroism
Tryptophan
Dimers
Labeling
Paramagnetic resonance
Digestion
Agglomeration
Aging of materials
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Deamidation, but not truncation, decreases the urea stability of a lens structural protein, βB1-crystallin. / Kim, Yung Hae; Kapfer, Deborah M.; Boekhorst, Jos; Lubsen, Nicolette H.; Bächinger, Hans Peter; Shearer, Thomas (Tom); David, Larry; Feix, Jimmy B.; Lampi, Kirsten.

In: Biochemistry, Vol. 41, No. 47, 26.11.2002, p. 14076-14084.

Research output: Contribution to journalArticle

Kim, Yung Hae ; Kapfer, Deborah M. ; Boekhorst, Jos ; Lubsen, Nicolette H. ; Bächinger, Hans Peter ; Shearer, Thomas (Tom) ; David, Larry ; Feix, Jimmy B. ; Lampi, Kirsten. / Deamidation, but not truncation, decreases the urea stability of a lens structural protein, βB1-crystallin. In: Biochemistry. 2002 ; Vol. 41, No. 47. pp. 14076-14084.
@article{c5f30ea3d9d2463db042964476d39d9d,
title = "Deamidation, but not truncation, decreases the urea stability of a lens structural protein, βB1-crystallin",
abstract = "Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type βb1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (βb1(ΔN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens βB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.",
author = "Kim, {Yung Hae} and Kapfer, {Deborah M.} and Jos Boekhorst and Lubsen, {Nicolette H.} and B{\"a}chinger, {Hans Peter} and Shearer, {Thomas (Tom)} and Larry David and Feix, {Jimmy B.} and Kirsten Lampi",
year = "2002",
month = "11",
day = "26",
doi = "10.1021/bi026288h",
language = "English (US)",
volume = "41",
pages = "14076--14084",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "47",

}

TY - JOUR

T1 - Deamidation, but not truncation, decreases the urea stability of a lens structural protein, βB1-crystallin

AU - Kim, Yung Hae

AU - Kapfer, Deborah M.

AU - Boekhorst, Jos

AU - Lubsen, Nicolette H.

AU - Bächinger, Hans Peter

AU - Shearer, Thomas (Tom)

AU - David, Larry

AU - Feix, Jimmy B.

AU - Lampi, Kirsten

PY - 2002/11/26

Y1 - 2002/11/26

N2 - Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type βb1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (βb1(ΔN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens βB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.

AB - Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type βb1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (βb1(ΔN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens βB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.

UR - http://www.scopus.com/inward/record.url?scp=0037180391&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037180391&partnerID=8YFLogxK

U2 - 10.1021/bi026288h

DO - 10.1021/bi026288h

M3 - Article

C2 - 12437365

AN - SCOPUS:0037180391

VL - 41

SP - 14076

EP - 14084

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 47

ER -