ARTICLE Amide temperature coefficients in the protein G B1 domain Jennifer H. Tomlinson • Mike P. Williamson Received: 13 September 2011 / Accepted: 6 October 2011 / Published online: 11 November 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Temperature coefficients have been measured for backbone amide 1 H and 15 N nuclei in the B1 domain of protein G (GB1), using temperatures in the range 283–313 K, and pH values from 2.0 to 9.0. Many nuclei display pH- dependent coefficients, which were fitted to one or two pK a values. 1 H coefficients showed the expected behaviour, in that hydrogen-bonded amides have less negative values, but for those amides involved in strong hydrogen bonds in regular secondary structure there is a negative correlation between strength of hydrogen bond and size of temperature coefficient. The best correlation to temperature coefficient is with secondary shift, indicative of a very approximately uniform thermal expansion. The largest pH-dependent changes in coefficient are for amides in loops adjacent to sidechain hydrogen bonds rather than the amides involved directly in hydrogen bonds, indicating that the biggest determinant of the temperature coefficient is temperature- dependent loss of structure, not hydrogen bonding. Amide 15 N coefficients have no clear relationship with structure. Keywords Temperature coefficient  Chemical shift  Hydrogen bond  pH  Protein G Introduction NMR as a structural method has a problem compared with X-ray crystallography, that there are far fewer experimental data measurements available for structure calculation. It is therefore important that NMR spectroscopists make the best use of the data that there are, particularly with the increasing emphasis on faster and more automated struc- ture calculations (Williamson and Craven 2009). A good example of this is the recent explosion of interest in chemical shifts, which has led to marked improvements in the speed and accuracy of structure calculations. Here, we look again at a parameter that has been studied for a long time, namely the temperature dependence of the chemical shift in amides in proteins, to define in more detail what its origins are, and whether it can provide useful structural information. Temperature coefficients were first studied in 1969 (Ohnishi and Urry 1969), when it was shown that the chemical shifts of amide protons involved in an intramo- lecular hydrogen bond change less with temperature than amide protons that are hydrogen bonded only with solvent. The measurement has been used extensively since then for identifying amides involved in hydrogen bonding, but mainly for peptides rather than proteins (reviewed in Williamson and Waltho 1992), because some of the tech- niques that have proved useful in proteins, such as solvent exchange rates of amide protons, are not accessible for peptides. The status of temperature coefficients was discussed in a landmark paper by Andersen et al. (1997), who showed that for many peptides, there is a strong correlation between the temperature coefficient and the secondary shift (the difference in chemical shift between its value in the peptide and in the random coil). The importance of this J. H. Tomlinson  M. P. Williamson (&) Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK e-mail: m.williamson@sheffield.ac.uk Present Address: J. H. Tomlinson Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK 123 J Biomol NMR (2012) 52:57–64 DOI 10.1007/s10858-011-9583-4