Carbenes Map Protein Surface | April 11, 2011 Issue - Vol. 89 Issue 15 | Chemical & Engineering News
Volume 89 Issue 15 | p. 10 | News of The Week
Issue Date: April 11, 2011

Carbenes Map Protein Surface

Structural Biology: Highly reactive species nonselectively label amino acids
Department: Science & Technology
Keywords: carbenes, proteins, topography, structure

Reactive carbenes provide a nonselective way to label amino acids on the surface of proteins, Canadian biochemists report (Anal. Chem., DOI: 10.1021/ac102655f). The method offers researchers a new tool for mapping protein topography that could lead to new insights into protein folding.


David C. Schriemer and Chanelle C. Jumper of the University of Calgary labeled proteins by mixing a diazirine-modified analog of the amino acid leucine with a protein of interest. Pulsed ultraviolet laser light converts this “photoleucine” into a carbene. Carbenes can insert into any X–H bond or add to any double bond in amino acids, but they are so short-lived that they can react only with accessible amino acids on the protein surface. The labeled protein, which has added mass, can then be detected mass spectrometrically.


Mass spectrometric analysis shows that each protein molecule on average acquires between one and four labels. Schriemer and Jumper plan to use tandem mass spectrometry to identify which amino acids are labeled.


High concentrations of the label are required to map the protein surface. Schriemer was pleased with how well the method worked because water can also react with the carbene, and this side reaction could have prevented proteins from being labeled at all.


Although the carbene itself reacts nondiscriminately with amino acids, the rest of the probe gives the researchers a handle for tailoring selectivity. The team hopes “to tune the specificity through additional moieties on the compound,” Schriemer says.


When he and Jumper used the carbene chemistry to label calmodulin, the labeling reflected changes in the protein’s accessible surface area caused by conformational changes in response to calcium binding.


“The authors hint that using commercially available UV lasers with nanosecond pulses along with the inherent nanosecond carbene lifetimes in solution should make submicrosecond labeling possible,” says Richard W. Vachet, a mass spectrometrist at the University of Massachusetts, Amherst. “If achieved, such fast labeling could provide access to new information about protein folding.”

 
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