Unbiased clustering of residues undergoing synchronous motions in proteins using NMR spin relaxation data.

Biological macromolecules are dynamic entities that transition between various conformational states, often playing a vital role in biological functions. Their inherent flexibility spans a broad range of timescales. Motions occurring within the microsecond to millisecond range are especially important, as they are integral to processes such as enzyme catalysis, folding, ligand binding, and allostery.

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.

Water irradiation devoid pulses enhance the sensitivity of H,H 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 [H,H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates H-detected spectra and minimize NOE intensity losses due to unwanted polarization exchange between water and labile protons.

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners.

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 H spectrum. These pulses were generated using a combination of an evolutionary algorithm and artificial intelligence.

PHRONESIS: A One-Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid-State NMR Spectroscopy.

Solid-state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane-bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance-specific assignments remain a bottleneck for this technique.

Is Disrupted Nucleotide-Substrate Cooperativity a Common Trait for Cushing's Syndrome Driving Mutations of Protein Kinase A?

Somatic mutations in the PRKACA gene encoding the catalytic α subunit of protein kinase A (PKA-C) are responsible for cortisol-producing adrenocortical adenomas. These benign neoplasms contribute to the development of Cushing's syndrome. The majority of these mutations occur at the interface between the two lobes of PKA-C and interfere with the enzyme's ability to recognize substrates and regulatory (R) subunits, leading to aberrant phosphorylation patterns and activation.

Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A.

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI.

T* weighted Deconvolution of NMR Spectra: Application to 2D Homonuclear MAS Solid-State NMR of Membrane Proteins.

2D homonuclear NMR spectroscopy is an essential technique to characterize small and large molecules, such as organic compounds, metabolites, and biomacromolecules at atomic resolution. However, for complex samples 2D homonuclear spectra display poor resolution, making spectral assignment very cumbersome. Here, we propose a new method that exploits the differential T* relaxation times of individual resonances and resolves the 2D NMR peaks into pseudo-3D spectra, where time is the 3 dimension.

Globally correlated conformational entropy underlies positive and negative cooperativity in a kinase's enzymatic cycle.

Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood.

Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy.

Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been challenging due to multiple samples required to characterize the hot spots at the protein interface. In this paper, we review our recently developed methods that greatly simplify PPI studies, which minimize the number of samples required to fully characterize residues involved in the protein-protein binding interface.

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.

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.

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.