Abstract
The efficiency of cell-free protein synthesis combined with combinatorial selective 15N-labelling provides a method for the rapid assignment of 15N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five 15N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the τ subunit of the Escherichia coli DNA polymerase III holoenzyme, τC16 and τC14. Since each of the five 15N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All 15N-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in τC16 vs. those in τC14, and the analysis of the structure and mobility of the C-terminal residues present in τC16 but not in τC14.
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Abbreviations
- τC14:
-
residues 499–625 of the τ subunit of E. coli DNA polymerase III with an additional N-terminal methionine
- τC16:
-
same as τC14, but including the C-terminal 18 residues 626–643 of τ
References
V. Dötsch G. Wagner (1996) J. Magn. Reson. B 111 310–313
S.D. Emerson R. Palermo C.M. Liu J.W. Tilley L. Chen W. Danho V.S. Madison D.N. Greeley G. Ju D.C. Fry (2003) Protein Sci. 12 811–822 Occurrence Handle10.1110/ps.0232803
D. Gao C.S. McHenry (2001) J. Biol. Chem. 276 4433–4440
K. Gehring E. Guittet (1995) J. Magn. Reson. B 109 206–208
T.D. Goddard D.G. Kneller (2004) SPARKY 3 University of California San Francisco
L. Guignard K. Ozawa S.E. Pursglove G. Otting N.E. Dixon (2002) FEBS Lett. 524 159–162 Occurrence Handle10.1016/S0014-5793(02)03048-X
T. Kigawa Y. Muto S. Yokoyama (1995) J. Biomol. NMR 6 129–134 Occurrence Handle10.1007/BF00211776
T. Kigawa T. Yabuki Y. Yoshida M. Tsutsui Y. Ito T. Shibata S. Yokoyama (1999) FEBS Lett. 442 15–19 Occurrence Handle10.1016/S0014-5793(98)01620-2
C. Klammt F. Löhr B. Schäfer W. Haase V. Dötsch H. Rüterjans C. Glaubitz F. Bernhard (2004) Eur. J. Biochem. 271 568–580 Occurrence Handle10.1111/j.1432-1033.2003.03959.x
E.H. Morita M. Shimizu T. Ogasawra Y. Endo R. Tanaka T. Kohno (2004) J. Biomol. NMR 30 37–45 Occurrence Handle10.1023/B:JNMR.0000042956.65678.b8
C. Neylon S.E. Brown A.V. Kralicek C.S. Miles C.A. Love N.E. Dixon (2000) Biochemistry 39 11989–11999 Occurrence Handle10.1021/bi001174w
K. Ozawa M.J. Headlam P.M. Schaeffer B.R. Henderson N.E. Dixon G. Otting (2004) Eur. J. Biochem. 271 4084–4093 Occurrence Handle10.1111/j.1432-1033.2004.04346.x
K. Ozawa N.E. Dixon G. Otting (2005a) IUBMB Life 57 615–622
K. Ozawa S. Jergic J.A. Crowther P.R. Thompson G. Wijffels G. Otting N.E. Dixon (2005b) J. Biomol. NMR 32 235–241 Occurrence Handle10.1007/s10858-005-7946-4
M.J. Parker M. Aulton-Jones A.M. Hounslow C.J. Craven (2004) J. Am. Chem. Soc. 126 5020–5021
M. Piotto V. Saudek V. Sklenář (1992) J. Biomol. NMR 2 661–665 Occurrence Handle10.1007/BF02192855
H. Ponstingl G. Otting (1998) J. Biomol. NMR 12 319–324 Occurrence Handle10.1023/A:1008293016073
P. Schmieder M. Leidert M.J.S. Kelly H. Oschkinat (1998) J. Magn. Reson. 131 199–201 Occurrence Handle10.1006/jmre.1997.1348
M. Schubert M. Smalla P. Schmieder H. Oschkinat (1999) J. Magn. Reson. 141 34–43 Occurrence Handle10.1006/jmre.1999.1881 Occurrence Handle1999JMagR.141...34S
M. Schubert H. Oschkinat P. Schmieder (2001a) J. Magn. Reson. 148 61–72 Occurrence Handle10.1006/jmre.2000.2222 Occurrence Handle2001JMagR.148...61S
M. Schubert H. Oschkinat P. Schmieder (2001b) J. Magn. Reson. 153 186–192 Occurrence Handle10.1006/jmre.2001.2447 Occurrence Handle2001JMagR.153..186S
M. Schubert H. Oschkinat P. Schmieder (2001c) J. Biomol. NMR 20 379–384 Occurrence Handle10.1023/A:1011206131623
M. Schubert D. Labudde D. Leitner H. Oschkinat P. Schmieder (2005) J. Biomol. NMR 31 115–127 Occurrence Handle10.1007/s10858-004-8263-z
J.X. Shi J.G. Pelton H.S. Cho D.E. Wemmer (2004) J. Biomol. NMR 28 235–247 Occurrence Handle10.1023/B:JNMR.0000013697.10256.74
D. Shortle (1994) J. Magn. Reson. B 105 88–90
M. Tashiro C.B. Rios G.T. Montelione (1995) J. Biomol. NMR 6 211–216 Occurrence Handle10.1007/BF00211785
N. Trbovic C. Klammt A. Koglin F. Löhr F. Bernhard V. Dötsch (2005) J. Am. Chem. Soc. 127 13504–13505 Occurrence Handle10.1021/ja0540270
P.S.C. Wu G. Otting (2005) J. Biomol. NMR 32 243–250 Occurrence Handle10.1007/s10858-005-8531-6
T. Yamazaki M. Yoshida S. Kanaya H. Nakamura K. Nagayama (1991) Biochemistry 30 6036–6047
T. Yamazaki S.M. Pascal A.U. Singer J.D. Forman-Kay L.E. Kay (1995) J. Am. Chem. Soc. 117 3556–3564
E.R. Zartler J. Hanson B.E. Jones A.D. Kiline G. Martin H. Mo M.J. Shapiro R. Wang H. Wu J. Yan (2003) J. Am. Chem. Soc. 125 10941–10946 Occurrence Handle10.1021/ja0348593
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Wu, P.S.C., Ozawa, K., Jergic, S. et al. Amino-acid Type Identification in 15N-HSQC Spectra by Combinatorial Selective 15N-labelling. J Biomol NMR 34, 13–21 (2006). https://doi.org/10.1007/s10858-005-5021-9
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DOI: https://doi.org/10.1007/s10858-005-5021-9