Abstract Multiwavelength anomalous dispersion (MAD) is the most widespread approach in structural biology to determine the crystal structure of a novel protein. Mass spectrometry is currently used to evaluate the selenomethionine (Se-Met) content in solution, but excluding fluorescence spectroscopy at the absorption edge, no other routine method to check the Se-Met incorporation and storage in the crystal state is yet available. Raman microscopy is an increasingly popular tool in physical biochemistry with applications ranging from studies on ligand binding to secondary structure analysis. Here we present a novel methodological development for the analysis, via Raman microscopy, of Se-Met labelled protein crystals to be used for MAD crystallography. The method is described and supported by validation and application to two novel proteins (a βγ-crystallin-like protein and a DNA-binding protein). Markers of the Se-Met residues are in the range from 570-600 cm-1 where proteins do not usually show Raman bands.

A novel method for detection of seleno-methionine incorporation in protein crystals via Raman microscopy

VERGARA, ALESSANDRO;MERLINO, ANTONELLO;PIZZO, ELIODORO;D'ALESSIO, GIUSEPPE;MAZZARELLA, LELIO
2008

Abstract

Abstract Multiwavelength anomalous dispersion (MAD) is the most widespread approach in structural biology to determine the crystal structure of a novel protein. Mass spectrometry is currently used to evaluate the selenomethionine (Se-Met) content in solution, but excluding fluorescence spectroscopy at the absorption edge, no other routine method to check the Se-Met incorporation and storage in the crystal state is yet available. Raman microscopy is an increasingly popular tool in physical biochemistry with applications ranging from studies on ligand binding to secondary structure analysis. Here we present a novel methodological development for the analysis, via Raman microscopy, of Se-Met labelled protein crystals to be used for MAD crystallography. The method is described and supported by validation and application to two novel proteins (a βγ-crystallin-like protein and a DNA-binding protein). Markers of the Se-Met residues are in the range from 570-600 cm-1 where proteins do not usually show Raman bands.
File in questo prodotto:
File Dimensione Formato  
ActaD_geodina.pdf

non disponibili

Tipologia: Altro materiale allegato
Licenza: Accesso privato/ristretto
Dimensione 195.43 kB
Formato Adobe PDF
195.43 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/205594
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 20
  • ???jsp.display-item.citation.isi??? 22
social impact