Untitled

Abstract

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.

Identification and Characterization of the Slowly Exchanging pH-dependent Conformational Rearrangement in KcsA

“…Unfortunately, the expression level of ⌬125-160 KcsA in D 2 O-based medium was quite low (less than 100 g liter Ϫ1 ). Therefore, we produced the protein in H 2 O-based medium supplemented with a deuterated amino acid mixture, as reported previously (28,29).…”

Variation in protein Cα‐related one‐bond J couplings

“…[58] Flavodoxin Recombinant D. vulgaris flavodoxin was prepared as described previously. [59] Measurements were carried out at 27…”

“…[58,73] 15 N-triply labeled DFPase. [58] Reference torsion-angle calculation from the 0.85-Å high-resolution PDB coordinates in dataset 1PJX ignored the numerous alternative atom records labeled B, C, or D. [74] No side-chain coordinates were given for Met1 and Glu2.…”

Extending the Size of Protein–RNA Complexes Studied by Nuclear Magnetic Resonance Spectroscopy

“…[20] This effects an average of 50-60 % fractional 2 Hlabeling, interestingly with a higher than average deuteration level for Ca carbons and a lower level for methyl groups. [17,21] This procedure is significantly cheaper than complete deuteration and does not require stepwise adaptation of the bacteria to the increased 2 H 2 O content, which can sometimes lead to slow cell growth, early stationary phase, or premature death of the culture. In the present study of the U2AF65/SF1/RNA complex, 100 % 2 H labeling was found to be crucial ( Figure 2).…”