Heteronuclear NMR Spectroscopy for Lysine NH₃ Groups in Proteins:  Unique Effect of Water Exchange on ¹⁵N Transverse Relaxation

Abstract

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH3 groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH3 groups, with 15N chemical shifts around approximately 33 ppm at pH 5.8 and 35 degrees C. Measurement of water-exchange rates and various types of 15N transverse relaxation rates for these NH3 groups, reveals that rapid water exchange dominates the 15N relaxation for antiphase coherence with respect to 1H through scalar relaxation of the second kind. As a consequence of this phenomenon, 15N line shapes of NH3 signals in a conventional 1H-15N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D 1H-15N correlation experiment that exclusively observes in-phase 15N transverse coherence (termed HISQC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t(1) (15N) time domain and as a result exhibits strikingly sharper 15N line shapes and higher intensities for NH3 cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between 15Nzeta and 13Cepsilon (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase 15N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH3 cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH3 groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH3 groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer.

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage

“…As direct detection of 15 N atoms suffers from low sensitivity and long relaxation times (30), 15 N Fig. 2A) (31). In accordance, in HSQC experiments of the purified ligation mixture we could not detect the 15 N amine signal using amine J H,N coupling constant ranging from 55 to 90 Hz (data not shown), although these covered the whole range of reported amine N correlations of the lysine amine group are often difficult to observe in HSQC experiments optimized for backbone amides, even when the water exchange rate is slow enough to permit their detection by 1 H NMR (31), for several reasons.…”

“…2B). As the radio frequency field strength that can be used for 15 N pulses is limited, application of a 15 N 180˚pulse at 115 ppm (close to the amide resonance frequency) leads to an imperfect pulse at 30 ppm (close to the lysine amine resonance frequency) and therefore to loss of signal intensity, and vice versa (31). With standard HSQC pulse programs it is therefore not possible to obtain maximal signal intensities for 15 N amide and 15 N amine groups simultaneously.…”

Probing Arginine Side‐Chains and Their Dynamics with Carbon‐Detected NMR Spectroscopy: Application to the 42 kDa Human Histone Deacetylase 8 at High pH

“…[3] Methods that probe large methyl-bearing side-chains have therefore been successfully applied to characterize the function of enzymes and macromolecular machines. [4] Techniques that probe charged side-chains are currently limited,[5] despite their importance for enzyme catalysis and protein–ligand interactions. With regard to probing arginine and lysine side-chains, conventional methods are often impeded by rapid exchange of the detected protons with the bulk solvent, which can lead to extensive line-broadening and effectively undetectable signals at and above physiological pH.…”

Internal Motions of Basic Side Chains of the Antennapedia Homeodomain in the Free and DNA-Bound States

“…Figure 2a shows the 1 H- 15 N heteronuclear in-phase single quantum coherence (HISQC) spectra 6 recorded for the Arg side-chain N ε -H ε moieties of the Antp homeodomain in the free and DNA-bound states at 25°C. Both samples were dissolved in a buffer of 20 mM potassium succinate (pH 5.8), 100 mM KCl, and 0.4 mM NaF (as a preservative).…”

“…( a ) Overlaid 1 H- 15 N HISQC spectra 6 recorded for the Arg side chains in the free protein (blue) and in the complex with 15-bp DNA (red). The region indicated by a dotted box is expanded in the inset.…”