Exploring protein interfaces with a general photochemical reagent

Autores
Gomez, Gabriela Elena; Cauerhff, Ana; Craig, Patricio Oliver; Goldbaum, Fernando Alberto; Delfino, Jose Maria
Año de publicación
2006
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. 3H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen eggwhite lysozyme(HEWL) and themonoclonal antibody IgG1D1.3. :CH2 labeling of free HEWL or complexed with IgG1 D1.3 yields 2.76 and 2.32 mmol CH 2 per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH 2. Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H15GLDNYR21, G117TDVQAWIR 125, and G22YSLGNWVCAAK33. Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.
Fil: Gomez, Gabriela Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina
Fil: Cauerhff, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Delfino, Jose Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina
Materia
Diazirine
Epitope Mapping
Hen Egg White Lysozyme
Methylene Carbene
Protein Complexes
Protein Footprinting
Protein Interfaces
Solvent-Accessible Surface Area
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/39137
Acceder
id CONICETDig_13a79c2959712c43cb750dd8053d888e
oai_identifier_str oai:ri.conicet.gov.ar:11336/39137
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Exploring protein interfaces with a general photochemical reagentGomez, Gabriela ElenaCauerhff, AnaCraig, Patricio OliverGoldbaum, Fernando AlbertoDelfino, Jose MariaDiazirineEpitope MappingHen Egg White LysozymeMethylene CarbeneProtein ComplexesProtein FootprintingProtein InterfacesSolvent-Accessible Surface Areahttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. 3H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen eggwhite lysozyme(HEWL) and themonoclonal antibody IgG1D1.3. :CH2 labeling of free HEWL or complexed with IgG1 D1.3 yields 2.76 and 2.32 mmol CH 2 per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH 2. Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H15GLDNYR21, G117TDVQAWIR 125, and G22YSLGNWVCAAK33. Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.Fil: Gomez, Gabriela Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Cauerhff, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Delfino, Jose Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaCold Spring Harbor Lab Press2006-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/39137Gomez, Gabriela Elena; Cauerhff, Ana; Craig, Patricio Oliver; Goldbaum, Fernando Alberto; Delfino, Jose Maria; Exploring protein interfaces with a general photochemical reagent; Cold Spring Harbor Lab Press; Protein Science; 15; 4; 4-2006; 744-7520961-83681469-896XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1110/ps.051960406/fullinfo:eu-repo/semantics/altIdentifier/doi/10.1110/ps.051960406info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:42:39Zoai:ri.conicet.gov.ar:11336/39137instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:42:39.868CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Exploring protein interfaces with a general photochemical reagent
title Exploring protein interfaces with a general photochemical reagent
spellingShingle Exploring protein interfaces with a general photochemical reagent
Gomez, Gabriela Elena
Diazirine
Epitope Mapping
Hen Egg White Lysozyme
Methylene Carbene
Protein Complexes
Protein Footprinting
Protein Interfaces
Solvent-Accessible Surface Area
title_short Exploring protein interfaces with a general photochemical reagent
title_full Exploring protein interfaces with a general photochemical reagent
title_fullStr Exploring protein interfaces with a general photochemical reagent
title_full_unstemmed Exploring protein interfaces with a general photochemical reagent
title_sort Exploring protein interfaces with a general photochemical reagent
dc.creator.none.fl_str_mv Gomez, Gabriela Elena
Cauerhff, Ana
Craig, Patricio Oliver
Goldbaum, Fernando Alberto
Delfino, Jose Maria
author Gomez, Gabriela Elena
author_facet Gomez, Gabriela Elena
Cauerhff, Ana
Craig, Patricio Oliver
Goldbaum, Fernando Alberto
Delfino, Jose Maria
author_role author
author2 Cauerhff, Ana
Craig, Patricio Oliver
Goldbaum, Fernando Alberto
Delfino, Jose Maria
author2_role author
author
author
author
dc.subject.none.fl_str_mv Diazirine
Epitope Mapping
Hen Egg White Lysozyme
Methylene Carbene
Protein Complexes
Protein Footprinting
Protein Interfaces
Solvent-Accessible Surface Area
topic Diazirine
Epitope Mapping
Hen Egg White Lysozyme
Methylene Carbene
Protein Complexes
Protein Footprinting
Protein Interfaces
Solvent-Accessible Surface Area
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. 3H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen eggwhite lysozyme(HEWL) and themonoclonal antibody IgG1D1.3. :CH2 labeling of free HEWL or complexed with IgG1 D1.3 yields 2.76 and 2.32 mmol CH 2 per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH 2. Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H15GLDNYR21, G117TDVQAWIR 125, and G22YSLGNWVCAAK33. Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.
Fil: Gomez, Gabriela Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina
Fil: Cauerhff, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Delfino, Jose Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina
description Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. 3H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen eggwhite lysozyme(HEWL) and themonoclonal antibody IgG1D1.3. :CH2 labeling of free HEWL or complexed with IgG1 D1.3 yields 2.76 and 2.32 mmol CH 2 per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH 2. Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H15GLDNYR21, G117TDVQAWIR 125, and G22YSLGNWVCAAK33. Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.
publishDate 2006
dc.date.none.fl_str_mv 2006-04
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/39137
Gomez, Gabriela Elena; Cauerhff, Ana; Craig, Patricio Oliver; Goldbaum, Fernando Alberto; Delfino, Jose Maria; Exploring protein interfaces with a general photochemical reagent; Cold Spring Harbor Lab Press; Protein Science; 15; 4; 4-2006; 744-752
0961-8368
1469-896X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/39137
identifier_str_mv Gomez, Gabriela Elena; Cauerhff, Ana; Craig, Patricio Oliver; Goldbaum, Fernando Alberto; Delfino, Jose Maria; Exploring protein interfaces with a general photochemical reagent; Cold Spring Harbor Lab Press; Protein Science; 15; 4; 4-2006; 744-752
0961-8368
1469-896X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1110/ps.051960406/full
info:eu-repo/semantics/altIdentifier/doi/10.1110/ps.051960406
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Cold Spring Harbor Lab Press
publisher.none.fl_str_mv Cold Spring Harbor Lab Press
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1844614459715223552
score 13.070432

Publicaciones similares