A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange

Journal of Biomolecular NMR - Tập 25 - Trang 291-311 - 2003
Frank Löhr1, Vicky Katsemi1, Judith Hartleib1, Ulrich Günther1, Heinz Rüterjans1
1Johann Wolfgang Goethe-University Frankfurt, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Frankfurt, Germany

Tóm tắt

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30–40 kDa to overcome problems arising from rapid 1H and 13C transverse relaxation. However, 1H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D2O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H2O. As a side-effect partial Cα protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary 1HN- and 1Hα-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D2O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of Cα protonation of individual amino acids is determined quantitatively and transverse relaxation properties of 1HN and 15N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.

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Thông tin
Thông tin xuất bản

Journal of Biomolecular NMR

Tập 25

291-311

Thông tin tác giả