TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor

Abstract

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.

13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1

“…In a preliminary attempt to improve spectral quality, we used a partially deuterated (∼80%) protein sample to reduce signal broadening due to transverse relaxation and obtained a 1 H- 13 C HSQC spectrum with modestly narrower linewidths and improved resolution (compare Figures 3b and 3d). We also attempted to utilize the methyl-TROSY effect (Kalverda et al 2014; Ollerenshaw et al 2003; Xu and Matthews 2013) by using the HMQC scheme. Preliminary HMQC tests run on a partially deuterated sample showed marginal improvements in linewidths for some peaks, but other peaks became less evident (Supplementary Figure S1).…”

NMR-Active Nuclei for Biological and Biomedical Applications

“…These can be affected by disease, drugs or environmental variation. The most common human body fluids to be analysed are blood (whole, plasma, serum) and urine [24,25] 2 H NMR has been in the study of water behaviour in bacterial spores, which can exhibit dormancy and thermal stability under extreme conditions. H spin pairs in the inhibitorcollagenase complex and also in the free inhibitor.…”

“…This property enables alignment of nuclei in an external magnetic field and absorption of radiofrequency radiation, which is the basis of the NMR experiment ( Figure 1). Nuclei that possess an even number of both neutrons and protons have no spin (spin quantum number = 0) and are not NMR-active, for example 2 He, 12 C, 16 O, 32 S. Nuclei for which the number of neutrons plus the number of protons is an odd number have a half-integer spin (i.e. spin quantum number = 1/2, 3/2, 5/2, 7/2, 9/2), for example 1 H, 13 C, 15 N, 19 F. Nuclei for which the number of neutrons and the number of protons are both odd have an integer spin (i.e.…”

“…spin quantum number = 1/2, 3/2, 5/2, 7/2, 9/2), for example 1 H, 13 C, 15 N, 19 F. Nuclei for which the number of neutrons and the number of protons are both odd have an integer spin (i.e. spin quantum number = 1, 2, 3), for example 2 H, 6 Li, 10 B, 14 N. In all nuclei for which the spin quantum number is greater than 1/2, the charge distribution of protons is asymmetric (Figure 2), which gives them an electric quadrupole moment in addition to their magnetic dipole moment. These 'quadrupolar nuclei', which constitute over two-thirds of all naturally occurring NMR-active nuclei, can have very short longitudinal relaxation times (T1) and produce broad NMR signals or none at all.…”

“…Exceptions include cases where samples have been prepared with various levels of deuteration in order to reduce or eliminate signals originating from background 1 H. In addition to removal of interfering 1 H signals from NMR spectra, deuteration also eliminates potential proton relaxation pathways and strong dipole-dipole interactions that would otherwise contribute to line broadening effects on the spectra. This is especially important when performing TROSY-type solution-state NMR experiments on detergent-solubilised membrane proteins where deuteration of both the protein and detergent may be essential [2][3][4]. An increasing number of studies have shown how sample deuteration and/or fast magic angle spinning (MAS) enable high-resolution protondetected solid-state NMR spectra to be obtained for samples of biological and biomedical origin.…”