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Efficient identification of amino acid types for fast protein backbone assignments
Authors:Horng D. Ou  Helen C. Lai  Zach Serber  Volker Dötsch
Affiliation:(1) Graduate Group in Biophysics, USA;(2) Departments of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology, University of California San Francisco, San Francisco, CA, 94143, U.S.A
Abstract:We describe a procedure that allows for very efficient identification of amino acid types in proteins by selective 15N-labeling. The usefulness of selective incorporation of 15N-labeled amino acids into proteins for the backbone assignment has been recognized for several years. However, widespread use of this method has been hindered by the need to purify each selectively labeled sample and by the relatively high cost of labeling with 15N-labeled amino acids. Here we demonstrate that purification of the selectively 15N-labeled samples is not necessary and that background-free HSQC spectra containing only the peaks of the overexpressed heterologous protein can be obtained in crude lysates from as little as 100 ml cultures, thus saving time and money. This method can be used for fast and automated backbone assignment of proteins.
Keywords:automated assignment  calmodulin  HSQC  SAM-domain  selective labeling
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