AI-designed NMR spectroscopy RF pulses for fast acquisition at high and ultra-high magnetic fields.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful high-resolution tool for characterizing biomacromolecular structure, dynamics, and interactions. However, the lengthy longitudinal relaxation of the nuclear spins significantly extends the total experimental time, especially at high and ultra-high magnetic field strengths. Although longitudinal relaxation-enhanced techniques have sped up data acquisition, their application has been limited by the chemical shift dispersion. Here we combined an evolutionary algorithm and artificial intelligence to design 1H and 15N radio frequency (RF) pulses with variable phase and amplitude that cover significantly broader bandwidths and allow for rapid data acquisition. We re-engineered the basic transverse relaxation optimized spectroscopy experiment and showed that the RF shapes enhance the spectral sensitivity of well-folded proteins up to 180 kDa molecular weight. These RF shapes can be tailored to re-design triple-resonance experiments for accelerating NMR spectroscopy of biomacromolecules at high fields.

Water irradiation devoid pulses enhance the sensitivity of 1H,1H nuclear Overhauser effects.

The nuclear Overhauser effect (NOE) is one of NMR spectroscopy's most important and versatile parameters. NOE is routinely utilized to determine the structures of medium-to-large size biomolecules and characterize protein-protein, protein-RNA, protein-DNA, and protein-ligand interactions in aqueous solutions. Typical [1H,1H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates 1H-detected spectra and minimize NOE intensity losses due to unwanted polarization exchange between water and labile protons. However, at high- and ultra-high magnetic fields, the excitation of the water signal during the execution of the NOESY pulse sequences may cause significant attenuation of NOE cross-peak intensities. Using an evolutionary algorithm coupled with artificial intelligence, we recently designed high-fidelity pulses [Water irrAdiation DEvoid (WADE) pulses] that elude water excitation and irradiate broader bandwidths relative to commonly used pulses. Here, we demonstrate that WADE pulses, implemented into the 2D [1H,1H] NOESY experiments, increase the intensity of the NOE cross-peaks for labile and, to a lesser extent, non-exchangeable protons. We applied the new 2D [1H,1H] WADE-NOESY pulse sequence to two well-folded, medium-size proteins, i.e., the K48C mutant of ubiquitin and the Raf kinase inhibitor protein. We observed a net increase of the NOE intensities varying from 30 to 170% compared to the commonly used NOESY experiments. The new WADE pulses can be easily engineered into 2D and 3D homo- and hetero-nuclear NOESY pulse sequences to boost their sensitivity.

Design and applications of water irradiation devoid RF pulses for ultra-high field biomolecular NMR spectroscopy.

Water suppression is of paramount importance for many biological and analytical NMR spectroscopy applications. Here, we report the design of a new set of binomial-like radio frequency (RF) pulses that elude water irradiation while exciting or refocusing the remainder of the 1H spectrum. These pulses were generated using a combination of an evolutionary algorithm and artificial intelligence. They display higher sensitivity relative to classical water suppression schemes and tunable water selectivity to avoid suppressing 1H resonances near the water signal. The broad bandwidth excitation obtained with these RF pulses makes them suitable for several NMR applications at high and ultra-high-field magnetic fields.

Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

Optimization of identity operation in NMR spectroscopy via genetic algorithm: Application to the TEDOR experiment.

Identity operation in the form of π pulses is widely used in NMR spectroscopy. For an isolated single spin system, a sequence of even number of π pulses performs an identity operation, leaving the spin state essentially unaltered. For multi-spin systems, trains of π pulses with appropriate phases and time delays modulate the spin Hamiltonian to perform operations such as decoupling and recoupling. However, experimental imperfections often jeopardize the outcome, leading to severe losses in sensitivity. Here, we demonstrate that a newly designed Genetic Algorithm (GA) is able to optimize a train of π pulses, resulting in a robust identity operation. As proof-of-concept, we optimized the recoupling sequence in the transferred-echo double-resonance (TEDOR) pulse sequence, a key experiment in biological magic angle spinning (MAS) solid-state NMR for measuring multiple carbon-nitrogen distances. The GA modified TEDOR (GMO-TEDOR) experiment with improved recoupling efficiency results in a net gain of sensitivity up to 28% as tested on a uniformly 13C, 15N labeled microcrystalline ubiquitin sample. The robust identity operation achieved via GA paves the way for the optimization of several other pulse sequences used for both solid- and liquid-state NMR used for decoupling, recoupling, and relaxation experiments.

Genetic algorithm optimized triply compensated pulses in NMR spectroscopy.

Sensitivity and resolution in NMR experiments are affected by magnetic field inhomogeneities (of both external and RF), errors in pulse calibration, and offset effects due to finite length of RF pulses. To remedy these problems, built-in compensation mechanisms for these experimental imperfections are often necessary. Here, we propose a new family of phase-modulated constant-amplitude broadband pulses with high compensation for RF inhomogeneity and heteronuclear coupling evolution. These pulses were optimized using a genetic algorithm (GA), which consists in a global optimization method inspired by Nature's evolutionary processes. The newly designed π and π/2 pulses belong to the 'type A' (or general rotors) symmetric composite pulses. These GA-optimized pulses are relatively short compared to other general rotors and can be used for excitation and inversion, as well as refocusing pulses in spin-echo experiments. The performance of the GA-optimized pulses was assessed in Magic Angle Spinning (MAS) solid-state NMR experiments using a crystalline U-(13)C, (15)N NAVL peptide as well as U-(13)C, (15)N microcrystalline ubiquitin. GA optimization of NMR pulse sequences opens a window for improving current experiments and designing new robust pulse sequences.

Fast and accurate quantification using Genetic Algorithm optimized 1H-13C refocused constant-time INEPT.

Using Genetic Algorithm, a global optimization method inspired by nature's evolutionary process, we have improved the quantitative refocused constant-time INEPT experiment (Q-INEPT-CT) of Mäkelä et al. (JMR 204 (2010) 124-130) with various optimization constraints. The improved 'average polarization transfer' and 'min-max difference' of new delay sets effectively reduces the experimental time by a factor of two (compared with Q-INEPT-CT, Mäkelä et al.) without compromising on accuracy. We also discuss a quantitative spectral editing technique based on average polarization transfer.