Abstract
A new protocol is described for the isotope (15N and 13C,15N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. 1H,13C and 15N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the Cγ resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.
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C. Bartels T.H. Xia M. Billeter P. Güntert K. Wüthrich (1995) J. Biomol. NMR 5 1–10 Occurrence Handle10.1007/BF00417486 Occurrence Handle7881269
M. Buck J. Boyd C. Redfield D.A. MacKenzie D.J. Jeenes D.B. Archer C.M. Dobson (1995) Biochemistry 34 4041–4055 Occurrence Handle10.1021/bi00012a023 Occurrence Handle7696270
M. Buck H. Schwalbe C.M. Dobson (1996) J. Mol. Biol. 257 669–683 Occurrence Handle10.1006/jmbi.1996.0193 Occurrence Handle8648632
R.T. Clubb V. Thanabal G. Wagner (1992) J. Magn. Reson. 97 213–217
L. Emsley G. Bodenhausen (1992) J. Magn. Reson. 97 135–148
B. Fischer (1996) Experientia Suppl. 75 143–161
Grimshaw, S. B. (1999) PhD Thesis, University of Oxford, United Kingdom
S. Grzesiek A. Bax (1992) J. Am. Chem. Soc. 114 6291–6293 Occurrence Handle10.1021/ja00042a003
S. Grzesiek A. Bax (1993) J. Biomol. NMR 3 185–204 Occurrence Handle8477186
R.L. Heinrikson (1971) J. Biol. Chem. 246 4090–4096 Occurrence Handle5105446
M. Hennig W. Bermel C.M. Dobson L.J. Smith H. Schwalbe (1999) J. Mol. Biol. 288 705–723 Occurrence Handle10.1006/jmbi.1999.2722 Occurrence Handle10329174
D.L. Hevehan E. Bernadez Clark ParticleDe (1997) Biotech. Bioeng. 54 221–230 Occurrence Handle10.1002/(SICI)1097-0290(19970505)54:3<221::AID-BIT3>3.0.CO;2-H
R. Keller (2004) The Computer Aided Resonance Assignment Tutorial CANTINA Verlag Goldau
J. Klein-Seetharaman M. Oikawa S.B. Grimshaw J. Wirmer E. Duchardt T. Ueda T. Imoto L.J. Smith C.M., Dobson H. Schwalbe (2002) Science 295 1719–1722 Occurrence Handle10.1126/science.1067680 Occurrence Handle11872841
F. Löhr V. Katsemi M. Betz J. Hartleib H. Rüterjans (2002) J. Biomol. NMR 22 153–164 Occurrence Handle10.1023/A:1014271204953 Occurrence Handle11883776
D.A. MacKenzie J.A. Spencer M.F. Gal-Coeffet ParticleLe D.B. Archer (1996) J. Biotechnol. 46 85–93 Occurrence Handle10.1016/0168-1656(95)00179-4 Occurrence Handle8672288
D. Marion M. Ikura R. Tschudin A.J. Bax (1989) J. Magn. Reson. 89 496–514
S. Mine T. Ueda Y. Hashimoto Y. Tanaka T. Imoto (1999) FEBS Lett. 448 33–37 Occurrence Handle10.1016/S0014-5793(99)00332-4 Occurrence Handle10217404
K.H. Mok P.J. Hore (2004) Methods 34 75–87 Occurrence Handle10.1016/j.ymeth.2004.03.006 Occurrence Handle15283917
G.T. Montelione B.A. Lyons S.D. Emerson M. Tashiro (1992) J. Am. Chem. Soc. 114 10974–10975 Occurrence Handle10.1021/ja00053a051
M. Piotto V. Saudek V. Sklenar (1992) J. Biomol. NMR 2 661–5 Occurrence Handle10.1007/BF02192855 Occurrence Handle1490109
C. Redfield C.M. Dobson (1988) Biochemistry 27 122–136 Occurrence Handle10.1021/bi00401a020 Occurrence Handle3349024
M. Sattler J. Schleucher C. Griesinger (1999) Progr. Nucl. Magn. Res. Spec. 34 93–158 Occurrence Handle10.1016/S0079-6565(98)00025-9
H. Schwalbe K.M. Fiebig M. Buck J.A. Jones S.B. Grimshaw A. Spencer S.J. Glaser L.J. Smith C.M. Dobson (1997) Biochemistry 36 8977–8991 Occurrence Handle10.1021/bi970049q Occurrence Handle9220986
H. Schwalbe S.B. Grimshaw A. Spencer M. Buck J. Boyd C.M. Dobson C. Redfield L.J. Smith (2001) Protein Sci. 10 677–688 Occurrence Handle10.1110/ps.43301 Occurrence Handle11274458
A.J. Shaka P.B. Barker R.J. Freeman (1985) J. Magn. Reson. 64 547–552
A.J. Shaka C.J. Lee A. Pines (1988) J. Magn. Reson. 77 274–293
C.M. Slupsky L.N. Gentile L.P. McIntosh (1998) Biochem. Cell Biol. 76 379–390 Occurrence Handle10.1139/bcb-76-2-3-379 Occurrence Handle9923707
L.J. Smith M.J. Sutcliffe C. Redfield C.M. Dobson (1991) Biochemistry 30 986–996 Occurrence Handle10.1021/bi00218a015 Occurrence Handle1989688
L.J. Smith M.J. Sutcliffe C. Redfield C.M. Dobson (1993) J. Mol. Biol. 229 930–944 Occurrence Handle10.1006/jmbi.1993.1097 Occurrence Handle8445657
A. Spencer L.A. Morozov-Roche W. Noppe D.A. MacKenzie D.J. Jeenes M. Joniau C.M. Dobson D.B. Archer (1999) Prot. Expr. Purif. 16 171–180 Occurrence Handle10.1006/prep.1999.1036
J. Wirmer C. Schlörb J. Klein-Seetharaman R. Hirano T. Ueda T. Imoto H. Schwalbe (2004) Angew Chem. Int. Ed. Engl. 43 5780–5785 Occurrence Handle10.1002/anie.200460907 Occurrence Handle15523735
D.S. Wishart C.G. Bigam J. Yao F. Abildgaard H.J. Dyson E. Oldfield J.L. Markley B.D. Sykes (1995) J. Biomol. NMR 6 135–140 Occurrence Handle10.1007/BF00211777 Occurrence Handle8589602
M. Wittekind L. Mueller (1993) J. Magn. Reson. 101 201–205
T. Yamazaki L.E. Kay (1993) J. Am. Chem. Soc. 115 11054–11055 Occurrence Handle10.1021/ja00076a099
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Schlörb, C., Ackermann, K., Richter, C. et al. Heterologous Expression of Hen Egg White Lysozyme and Resonance Assignment of Tryptophan Side Chains in its Non-native States. J Biomol NMR 33, 95–104 (2005). https://doi.org/10.1007/s10858-005-2063-y
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DOI: https://doi.org/10.1007/s10858-005-2063-y