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 Se-Met content in solution, but a routine method to check the Se-Met inclusion and storage in the crystal state is not yet available. Raman microscopy is having increasing application into molecular biology, ranging from studies on ligand binding [1] and secondary structure analysis [2]. Here we present a novel tried-and tested methodological development to conduct, via Raman microscopy, analysis on Se-Met labelled protein crystals to be used for MAD crystallography. The method is described, and is 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 usually do not show any Raman band. [1] P. R.Carey, J. Dong, Biochemistry 2004, 43, 8885-8893. [2] P. R. Carey, Ann. Rev. Phys. Chem. 2006, 57, 527-554.

A novel method for detection of Se-Met inclusion into protein crystals via Raman microscopy

VERGARA, ALESSANDRO;MERLINO, ANTONELLO;PIZZO, ELIODORO;MAZZARELLA, LELIO
2007

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 Se-Met content in solution, but a routine method to check the Se-Met inclusion and storage in the crystal state is not yet available. Raman microscopy is having increasing application into molecular biology, ranging from studies on ligand binding [1] and secondary structure analysis [2]. Here we present a novel tried-and tested methodological development to conduct, via Raman microscopy, analysis on Se-Met labelled protein crystals to be used for MAD crystallography. The method is described, and is 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 usually do not show any Raman band. [1] P. R.Carey, J. Dong, Biochemistry 2004, 43, 8885-8893. [2] P. R. Carey, Ann. Rev. Phys. Chem. 2006, 57, 527-554.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/307830
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