A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange.
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 (1)H and (13)C transverse relaxation. However, (1)H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D(2)O-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% H(2)O. As a side-effect partial C(alpha) protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary (1)H(N)- and (1)H(alpha)-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D(2)O 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 C(alpha) protonation of individual amino acids is determined quantitatively and transverse relaxation properties of (1)H(N) and (15)N 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.
Löhr
Frank
F
Johann Wolfgang Goethe-University Frankfurt, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Marie Curie-Strasse 9, N230, 1.OG, D-60439 Frankfurt, Germany. murph@bpc.uni-frankfurt.de
Katsemi
Vicky
V
Hartleib
Judith
J
Günther
Ulrich
U
Rüterjans
Heinz
H
D000577:Amides; D000595:Amino Acid Sequence; D000818:Animals; D049832:Decapodiformes / Q000737:chemistry; D003903:Deuterium; D041961:Deuterium Exchange Measurement; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
D000577:Amides; D011506:Proteins; D003903:Deuterium; D044345:Phosphoric Triester Hydrolases; C020061:diisopropyl-fluorophosphatase