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1.
Protein Sci ; 16(7): 1464-78, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17567739

RESUMEN

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 3,3'-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 degrees 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.


Asunto(s)
Cloroplastos/metabolismo , Citrato (si)-Sintasa/metabolismo , Proteínas de Choque Térmico Pequeñas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Citrato (si)-Sintasa/química , Dimerización , Proteínas de Choque Térmico Pequeñas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos
2.
Proteins ; 62(4): 1044-52, 2006 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-16385579

RESUMEN

Crosslinking of small heat-shock proteins (sHsps) by tissue transglutaminase (tTG) is enhanced by stress and under pathological conditions. We here used hexapeptide probes to determine the amine donor (K) and acceptor (Q) sites for tTG in Hsp20. Mass spectrometric peptide mass fingerprinting and peptide fragmentation established that Q31 and the C-terminal K162 are involved in inter- and intramolecular crosslinking (transamidation). Q31 is a conserved glutamine in sHsps where the neighboring residue determines its reactivity. Moreover, we detected highly efficient simultaneous deamidation of Q66, which suggests that tTG-catalyzed transamidation and deamidation is specific for different glutamine residues.


Asunto(s)
Proteínas del Choque Térmico HSP20/metabolismo , Transglutaminasas/metabolismo , Amidas/metabolismo , Clonación Molecular , Escherichia coli , Proteínas de Unión al GTP , Células HeLa , Humanos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteínas Recombinantes/metabolismo , Transfección
3.
J Biomol Tech ; 16(3): 197-208, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16461943

RESUMEN

Detection of posttranslational modifications is expected to be one of the major future experimental challenges for proteomics. We describe herein a mass spectrometric procedure to screen for protein modifications by peptide mass fingerprinting that is based on post-data acquisition improvement of the mass accuracy by exporting the peptide mass values into analytical software for multipoint recalibration on recognized peaks. Subsequently, the calibrated peak mass data set is used in searching for modified peptides, i.e., peptides possessing specific mass deviations. In order to identify the location of Lys- and Gln-residues available for transglutaminase-catalyzed isopeptide bond formation, mammalian small heat shock proteins (sHsps) were screened for labeling with the two hexapeptide probes GQDPVR and GNDPVK in presence of transglutaminase. Peptide modification due to cross-linking of the GQDPVR hexa-peptide probe was detected for C-terminal Lys residues. Novel transglutaminase-susceptible Gln sites were identified in two sHsps (Q31/Q27 in Hsp20 and HspB2, respectively), by cross-linking of the GNDPVK hexapeptide probe. Deamidation of specific Gln residues was also detected, as well an isopeptide derived from intramolecular Gln-Lys isopeptide bond formation. We conclude that peptide mass fingerprinting can be an efficient way of screening for various posttranslational modifications. Basically any instrumentation for MALDI mass spectrometry can be used, provided that post-data acquisition recalibration is applied.


Asunto(s)
Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normas , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Calibración , Catálisis , Datos de Secuencia Molecular
4.
Extremophiles ; 11(5): 659-66, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17486291

RESUMEN

Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889). Strong binding of sHsps was detected to a peptide corresponding to the most N-terminal alpha-helix in CS (amino acids Leu(13) to Gln(27)). The N-terminal alpha-helices in the CS dimer intertwine with the C-terminus in the other subunit and together form a stem-like structure which is protruding from the CS dimer. This stem-like structure is absent in thermostable forms of CS from thermophilic archaebacteria like Pyrococcus furiosus and Sulfolobus solfatacarium. These data therefore suggest that thermostabilization of thermosensitive CS by sHsps is achieved by stabilization of the C- and N-terminae in the protruding thermosensitive softspot, which is absent in thermostable forms of the CS dimer.


Asunto(s)
Citrato (si)-Sintasa/metabolismo , Proteínas de Choque Térmico Pequeñas/metabolismo , Temperatura , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Citrato (si)-Sintasa/química , Dimerización , Estabilidad de Enzimas , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequeñas/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Porcinos , Cadena B de alfa-Cristalina/metabolismo
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