Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase

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Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase. / Åhrman, Emma; Lambert, Wietske; Aquilina, J. Andrew; Robinson, Carol V.; Emanuelsson, Cecilia.

In: Protein Science, Vol. 16, No. 7, 2007, p. 1464-1478.

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Åhrman, Emma ; Lambert, Wietske ; Aquilina, J. Andrew ; Robinson, Carol V. ; Emanuelsson, Cecilia. / Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase. In: Protein Science. 2007 ; Vol. 16, No. 7. pp. 1464-1478.

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TY - JOUR

T1 - Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase

AU - Åhrman, Emma

AU - Lambert, Wietske

AU - Aquilina, J. Andrew

AU - Robinson, Carol V.

AU - Emanuelsson, Cecilia

PY - 2007

Y1 - 2007

N2 - The molecular mechanism whereby the small heat-shock protein ( sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase ( CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross- linker captured Hsp21 in dodecameric form, even at increased temperature ( 47 C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.

AB - The molecular mechanism whereby the small heat-shock protein ( sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase ( CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross- linker captured Hsp21 in dodecameric form, even at increased temperature ( 47 C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.

KW - heat-shock protein

KW - small

KW - chemical cross-linking

KW - mass spectrometric peptide mapping

KW - protein-protein interactions

KW - citrate synthase

U2 - 10.1110/ps.072831607

DO - 10.1110/ps.072831607

M3 - Article

VL - 16

SP - 1464

EP - 1478

JO - Protein Science

JF - Protein Science

SN - 1469-896X

IS - 7

ER -