Photo-CIDNP NMR spectroscopy of a heme-containing protein

There are relatively few examples of the application of photo-CIDNP NMR spectroscopy to chromophore-containing proteins. The most likely reason for this is that simultaneous absorption of light by the photosensitiser molecule and the protein chromophore reduces the effectiveness of the photochemical reaction that produces the observed nuclear polarisation. We present details of experiments performed on the air-oxidised form of a small cytochrome, from the thermophilic bacterium Hydrogenobacter thermophilus, using both the wild-type protein and apo and holo forms of a double alanine b-type mutant. We show that, along with the apo state, it is possible to generate CIDNP in the air-oxidised form of the b-type mutant, but not in the corresponding c-type cytochrome. This finding is supported by control experiments using horse-heart cytochrome c.     

EPIC- and CHANCE-HSQC: Two N-photo-CIDNP-enhanced pulse sequences for the sensitive detection of solvent-exposed tryptophan

Photochemically induced dynamic nuclear polarization (photo-CIDNP) of nuclei other than ¹H offers a tremendous potential for sensitivity enhancement in liquid state NMR under mild, physiologically relevant conditions. Photo-CIDNP enhancements of ¹⁵N magnetization are much larger than those typically observed for ¹H. However, the low gyromagnetic ratio of ¹⁵N prevents a full fruition of the potential signal-to-noise gains attainable via ¹⁵N photo-CIDNP. Here, we propose two novel pulse sequences, EPIC- and CHANCE-HSQC, tailored to overcome the above limitation. EPIC-HSQC exploits the strong ¹H polarization and its subsequent transfer to non-equilibrium N_z magnetization prior to ¹⁵N photo-CIDNP laser irradiation. CHANCE-HSQC synergistically combines ¹H and ¹⁵N photo-CIDNP. The above pulse sequences, tested on tryptophan (Trp) and the Trp-containing protein apoHmpH, were found to display up to 2-fold higher sensitivity than the reference NPE-SE-HSQC pulse train (based on simple ¹⁵N photo-CIDNP followed by N–H polarization transfer), and up to a ca. 3-fold increase in sensitivity over the corresponding dark pulse schemes (lacking laser irradiation). The observed effects are consistent with the predictions from a theoretical model of photo-CIDNP and prove the potential of ¹⁵N and ¹H photo-CIDNP in liquid state heteronuclear correlation NMR.     

Fragment Screening and Fast Micromolar Detection on a Benchtop NMR Spectrometer Boosted by Photoinduced Hyperpolarization

Fragment-based drug design is a well-established strategy for rational drug design, with nuclear magnetic resonance (NMR) on high-field spectrometers as the method of reference for screening and hit validation. However, high-field NMR spectrometers are not only expensive, but require specialized maintenance, dedicated space, and depend on liquid helium cooling which became critical over the recurring global helium shortages. We propose an alternative to high-field NMR screening by applying the recently developed approach of fragment screening by photoinduced hyperpolarized NMR on a cryogen-free 80 MHz benchtop NMR spectrometer yielding signal enhancements of up to three orders in magnitude. It is demonstrated that it is possible to discover new hits and kick-off drug design using a benchtop NMR spectrometer at low micromolar concentrations of both protein and ligand. The approach presented performs at higher speed than state-of-the-art high-field NMR approaches while exhibiting a limit of detection in the nanomolar range. Photoinduced hyperpolarization is known to be inexpensive and simple to be implemented, which aligns greatly with the philosophy of benchtop NMR spectrometers. These findings open the way for the use of benchtop NMR in near-physiological conditions for drug design and further life science applications.     

固态Photo-CIDNP效应

光化学诱导动态核极化(photo-CIDNP)是一种在光照条件下由于产生非玻尔兹曼核自旋极化而使核磁共振(NMR)波谱信号强度发生明显变化的效应。这种效应在液体NMR中已为人所熟知,并通过经典的自由基对机理得到解释。固态photo-CIDNP效应发现的较晚,本文介绍了在光合反应中心及蓝光受体中发现的固态photo-CIDNP效应,详细阐述了固态photo-CIDNP效应产生的自由基对自旋动力学的机理,包括三旋混合(TSM)、衰变差异(DD)和弛豫差异(DR),重点介绍了类球红杆菌光合反应中心固态photo-CIDNP效应的磁场依赖性,这种场依赖性在同一分子中的不同核之间表现出明显的差异。本文综述了固态photo-CIDNP效应的现象、理论及其磁场依赖特性的最新进展。