NMR detection of bifurcated hydrogen bonds in large proteins.

Hydrogen bonds play critical roles in protein structure, stability, and function. Most hydrogen bonds in proteins are derived from their crystal structures and the use of standard covalent geometry information, because the positions of hydrogen atoms are defined only in a limited number of ultrahigh-resolution crystal structures. On the other hand, NMR structure calculations rely mostly on the distances between hydrogen atoms, and the positions of the heavy atoms involved in hydrogen bonds are defined by standard covalent geometry information. Consequently, NMR structures do not contain independent information about hydrogen bonds. One of the most important advances in NMR spectroscopy in the past 10 years was the direct detection of hydrogen bonds through trans-hydrogen-bond scalar couplings.1 However, trans-hydrogenbond scalar couplings are generally small so that they cannot be measured for large proteins. No scalar coupling constants for hydrogen bonds involving water molecules have been measured. Unlike the electron-mediating scalar couplings, isotope effects are vibrational phenomena in nature and are propagated through either covalent bonds or hydrogen bonds and potentially can be used as an alternative approach for the direct detection of hydrogen bonds. A wealth of literatures has documented the usefulness of the isotope effects in NMR analysis of small compounds,2 small proteins,3 and nucleic acids.4 Trans-hydrogen-bond deuterium isotope effects on the chemical shifts of nucleic acids have been reported,4 but no such effects have been reported on proteins. In this communication, we report the first observation of transhydrogen-bond two-bond H/D isotope effects, 2h∆1H, and transhydrogen-bond three-bond isotope effects, 3h∆15N, in a large protein using the recently developed isotopomer-selective (IS) TROSY technique5 and show that such deuterium isotope effects can be used to detect a most common type of bifurcated hydrogen bonds in which a heavy atom, usually oxygen, is involved in two hydrogen bonds. The protein used for this study was yeast cytosine deaminase (yCD), a 35 kDa homodimeric enzyme that catalyzes the deamination of not only the physiological substrate cytosine but also the prodrug 5-fluorocytosine and is widely used for gene-directed enzyme/prodrug therapy for the treatment of cancer.6 Two highresolution crystal structures have been reported for yCD in complex with the transition state analogue 2-pyrimidinone (Py) for the deamination of cytosine, one at 1.14 Å7a and the other at 1.6 Å resolution.7b We have been studying the catalytic mechanism of yCD and its role in the activation of the prodrug 5-fluorocytosine by biochemical, NMR, and computational methods.5,8 Figure 1 shows expanded regions of the 15N-1H IS-TROSY spectrum of yCD in complex with the transition state analogue 5-fluoro-2-pyrimidinone (5FPy) in either 50% H2O/50% D2O (mixture solvent sample) or 95% H2O/5% D2O (water sample), recorded on a 900 MHz NMR spectrometer. The figure contains the side-chain amide resonances of Asn51 and both backbone and side-chain amide resonances of Asn113. Each side-chain amide has two hydrogen atoms, one in the trans (E) and the other in the cis (Z) configuration with respect to the carboxamide oxygen. In the mixture solvent, the relative population of each of the four possible isotopomers, NHEHZ, NHEDZ, NDEHZ, and NDEDZ, is about equal if D/H isotope fractionation factors are close to one. The IS-TROSY experiment exclusively detects the resonances of semideuterated † Department of Biochemistry and Molecular Biology. ‡ Department of Chemistry. § Present address: Laboratory of Chemical Physics, NIDDK National Institute of Health, Bethesda, MD 20892, USA. Figure 1. Regions of 2D 15N-1H IS-TROSY spectra of [U-2H,13C,15N]yCD recorded on a Bruker AVANCE 900 MHz NMR spectrometer equipped with a cryoprobe at 25 °C. Panels (a) and (c) are from the spectrum obtained with a yCD sample in 50% H2O/50% D2O, and (b) and (d) are corresponding regions with a sample in 95% H2O/5% D2O. The hydrogenbonding network involving Asn51 and Asn113 are illustrated on top with distances between heavy atoms (Å) indicated for each hydrogen bond. NMR samples were made in 100 mM potassium phosphate buffer, pH 7.0 (pH meter reading), with 100 μM NaN3 and 20 μM DSS (an internal NMR reference). The samples contained ∼1.5 mM yCD (protomer concentration) and 20 mM the transition state analogue 5FPy. NMR assignment was achieved with IS-TROSY techniques and reported earlier.5 The resonance marked with the asterisk in (d) is a folded peak from arginine side chains. The spectrum for panels (a) and (c) was recorded with 16 scans and a 2 s delay time, t1max(N) ) 53 ms and t2max(H) ) 285 ms, resulting in the experimental time of 3.5 h, and the spectrum for panels (b) and (d) was recorded and processed in the same manner, but with 64 scans, resulting in an experimental time of 13.9 h. Published on Web 02/05/2008