In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT*6 and TPMT*8.

Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 µM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups.

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for (1)H spins in methyl (13)CHD2 groups, which improves the characterization of fast exchange processes. The influence of (1)H-(1)H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R 1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different (1)H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any (1)H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by (1)H and (13)C CPMG experiments. The present R 1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.

Isotope labeling methods for large systems.

A major drawback of nuclear magnetic resonance (NMR) spectroscopy compared to other methods is that the technique has been limited to relatively small molecules. However, in the last two decades the size limit has been pushed upwards considerably and it is now possible to use NMR spectroscopy for structure calculations of proteins of molecular weights approaching 100 kDa and to probe dynamics for supramolecular complexes of molecular weights in excess of 500 kDa. Instrumental for this progress has been development in instrumentation and pulse sequence design but also improved isotopic labeling schemes that lead to increased sensitivity as well as improved spectral resolution and simplification. These are described and discussed in this chapter, focusing on labeling schemes for amide proton and methyl proton detected experiments. We also discuss labeling methods for other potentially useful positions in proteins.

Isotope labeling methods for relaxation measurements.

Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynamics. Not only is it possible to use it for determination of time constant(s) for molecular reorientation but it can also be used to characterize internal motions on time scales from picoseconds to seconds. Traditionally, uniformly (15)N labeled samples have been used for these experiments but it is clear that this limits the applications. For instance, sensitivity for large systems is dramatically increased if dynamics is probed at methyl groups and structural characterization of low-populated states requires measurements on (13)Cα, (13)Cß or (13)CO or (1)Hα. Unfortunately, homonuclear scalar couplings may lead to artifacts in the latter types of experiments and selective isotopic labeling schemes that only label the desired position are necessary. Both selective and uniform labeling schemes for measurements of relaxation rates for a large number of positions in proteins are discussed in this chapter.