caa a22 4500 445325003 CHVBK 20180317142717.0 cr unu---uuuuu 170323e20110101xx s 000 0 eng 10.1007/s10858-010-9459-z doi (NATIONALLICENCE)springer-10.1007/s10858-010-9459-z Amino acid selective unlabeling for sequence specific resonance assignments in proteins [Elektronische Daten] [B. Krishnarjuna, Garima Jaipuria, Anushikha Thakur, Patrick D'Silva, Hanudatta Atreya] Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly 13C/15N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {12COi-15Ni+1}-filtered HSQC, which aids in linking the 1HN/15N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i+1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i−1, i and i+1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to 2H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of 14N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies. The Author(s), 2010 Selective unlabeling nationallicence Sequence specific resonance assignment nationallicence Isotope labeling nationallicence Protein structure nationallicence Krishnarjuna B. NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut Jaipuria Garima NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut Thakur Anushikha NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut D'Silva Patrick Department of Biochemistry, Indian Institute of Science, 560012, Bangalore, India aut Atreya Hanudatta NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut Journal of Biomolecular NMR Springer Netherlands 49/1(2011-01-01), 39-51 0925-2738 49:1<39 2011 49 10858 https://doi.org/10.1007/s10858-010-9459-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-010-9459-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Krishnarjuna B. NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut NATIONALLICENCE 700 1- Jaipuria Garima NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut NATIONALLICENCE 700 1- Thakur Anushikha NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut NATIONALLICENCE 700 1- D'Silva Patrick Department of Biochemistry, Indian Institute of Science, 560012, Bangalore, India aut NATIONALLICENCE 700 1- Atreya Hanudatta NMR Research Centre, Indian Institute of Science, 560012, Bangalore, India aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 49/1(2011-01-01), 39-51 0925-2738 49:1<39 2011 49 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer