Slice & Dice: nested spin-lattice relaxation measurements.

Spin-lattice relaxation rate (R1) measurements are commonly used to characterize protein dynamics. However, the time needed to collect the data can be quite long due to long relaxation times of the low-gamma nuclei, especially in the solid state. We present a method to collect backbone heavy atom relaxation data by nesting the collection of datasets in the solid state. This method results in a factor of 2 to 2.5 times faster data acquisition for backbone R1 relaxation data for the 13C and 15N sites of proteins.

Nuclear spin diffusion under fast magic-angle spinning in solid-state NMR.

Solid-state nuclear spin diffusion is the coherent and reversible process through which spin order is transferred via dipolar couplings. With the recent increases in magic-angle spinning (MAS) frequencies and magnetic fields becoming routinely applied in solid-state nuclear magnetic resonance, understanding how the increased 1H resolution obtained affects spin diffusion is necessary for interpretation of several common experiments. To investigate the coherent contributions to spin diffusion with fast MAS, we have developed a low-order correlation in Liouville space model based on the work of Dumez et al. (J. Chem. Phys. 33, 224501, 2010). Specifically, we introduce a new method for basis set selection, which accounts for the resonance-offset dependence at fast MAS. Furthermore, we consider the necessity of including chemical shift, both isotropic and anisotropic, in the modeling of spin diffusion. Using this model, we explore how different experimental factors change the nature of spin diffusion. Then, we show case studies to exemplify the issues that arise in using spin diffusion techniques at fast spinning. We show that the efficiency of polarization transfer via spin diffusion occurring within a deuterated and 100% back-exchanged protein sample at 60 kHz MAS is almost entirely dependent on resonance offset. We additionally identify temperature-dependent magnetization transfer in beta-aspartyl L-alanine, which could be explained by the influence of an incoherent relaxation-based nuclear Overhauser effect.

Optimisation of 1H PMLG homonuclear decoupling at 60 kHz MAS to enable 15N-1H through-bond heteronuclear correlation solid-state NMR spectroscopy.

The Lee-Goldburg condition for homonuclear decoupling in 1H magic-angle spinning (MAS) solid-state NMR sets the angle θ, corresponding to arctan of the ratio of the rf nutation frequency, ν1, to the rf offset, to be the magic angle, θm, equal to tan-1(√2) = 54.7°. At 60 kHz MAS, we report enhanced decoupling compared to MAS alone in a 1H spectrum of 15N-glycine with at θ = 30° for a ν1 of ∼100 kHz at a 1H Larmor frequency, ν0, of 500 MHz and 1 GHz, corresponding to a high chemical shift scaling factor (λCS) of 0.82. At 1 GHz, we also demonstrate enhanced decoupling compared to 60 kHz MAS alone for a lower ν1 of 51 kHz, i.e., a case where the nutation frequency is less than the MAS frequency, with θ = 18°, λCS = 0.92. The ratio of the rotor period to the decoupling cycle time, Ψ = τr/τc, is in the range 0.53 to 0.61. Windowed decoupling using the optimised parameters for a ν1 of ∼100 kHz also gives good performance in a 1H spin-echo experiment, enabling implementation in a 1H-detected 15N-1H cross polarisation (CP)-refocused INEPT heteronuclear correlation NMR experiment. Specifically, initial 15N transverse magnetisation as generated by 1H-15N CP is transferred back to 1H using a refocused INEPT pulse sequence employing windowed 1H decoupling. Such an approach ensures the observation of through-bond N-H connectivities. For 15N-glycine, while the CP-refocused INEPT experiment has a lower sensitivity (∼50%) as compared to a double CP experiment (with a 200 µs 15N to 1H CP contact time), there is selectivity for the directly bonded NH3+ moiety, while intensity is observed for the CH21H resonances in the double CP experiment. Two-dimensional 15N-1H correlation MAS NMR spectra are presented for the dipeptide ß-AspAla and the pharmaceutical cimetidine at 60 kHz MAS, both at natural isotopic abundance. For the dipeptide ß-AspAla, different build-up dependence on the first spin-echo duration is observed for the NH and NH3+ moieties demonstrating that the experiment could be used to distinguish resonances for different NHx groups.

Correction: Optimisation of 1H PMLG homonuclear decoupling at 60 kHz MAS to enable 15N-1H through-bond heteronuclear correlation solid-state NMR spectroscopy.

Correction for 'Optimisation of 1H PMLG homonuclear decoupling at 60 kHz MAS to enable 15N-1H through-bond heteronuclear correlation solid-state NMR spectroscopy' by Jacqueline Tognetti et al., Phys. Chem. Chem. Phys., 2022, 24, 20258-20273, https://doi.org/10.1039/D2CP01041K.

Docking domain-mediated subunit interactions in natural product megasynth(et)ases.

Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) multienzymes produce numerous high value metabolites. The protein subunits which constitute these megasynth(et)ases must undergo ordered self-assembly to ensure correct organisation of catalytic domains for the biosynthesis of a given natural product. Short amino acid regions at the N- and C-termini of each subunit, termed docking domains (DDs), often occur in complementary pairs, which interact to facilitate substrate transfer and maintain pathway fidelity. This review details all structurally characterised examples of NRPS and PKS DDs to date and summarises efforts to utilise DDs for the engineering of biosynthetic pathways.

Mechanism of intersubunit ketosynthase-dehydratase interaction in polyketide synthases.

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity.

Revealing Intermolecular Hydrogen Bonding Structure and Dynamics in a Deep Eutectic Pharmaceutical by Magic-Angle Spinning NMR Spectroscopy.

Liquid forms of pharmaceuticals (ionic liquids and deep eutectic solvents) offer a number of potential advantages over solid-state drugs; a key question is the role of intermolecular hydrogen bonding interactions in enabling membrane transport. Characterization is challenging since high sample viscosities, typical of liquid pharmaceutical formulations, hamper the use of conventional solution NMR at ambient temperature. Here, we report the application of magic-angle spinning (MAS) NMR spectroscopy to the deep eutectic pharmaceutical, lidocaine ibuprofen. Using variable temperature MAS NMR, the neat system, at a fixed molar ratio, can be studied over a wide range of temperatures, characterized by changing mobility, using a single experimental setup. Specific intermolecular hydrogen bonding interactions are identified by two-dimensional 1H-1H NOESY and ROESY MAS NMR experiments. Hydrogen-bonding dynamics are quantitatively determined by following the chemical exchange process between the labile protons by means of line-width analysis of variable temperature 1H MAS NMR spectra.

Binding of Distinct Substrate Conformations Enables Hydroxylation of Remote Sites in Thaxtomin D by Cytochrome P450 TxtC.

Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation, and natural product biosynthesis. Here we report biochemical, structural, and theoretical studies of TxtC, an unusual bifunctional CYP involved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites within the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the α-carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionalization, X-ray crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density corresponding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-intermediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen complex into the reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the π-face of the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second, energetically similar but conformationally distinct binding mode in which the α-hydrogen of the Phe residue is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the α-hydrogen is sufficiently close to the ferryl oxygen atom to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and monohydroxylated intermediate.

Modulation of Transmembrane Domain Interactions in Neu Receptor Tyrosine Kinase by Membrane Fluidity and Cholesterol.

The activation mechanism of the ErbB family of receptors is of considerable medical interest as they are linked to a number of human cancers, including an aggressive form of breast cancer. In the rat analogue of the human ErbB2 receptor, referred to as Neu, a point mutation in the transmembrane domain (V664E) has been shown to trigger oncogenic transformation. While the structural impact of this mutation has been widely studied in the past to yield models for the active state of the Neu receptor, little is known about the impact of cholesterol on its structure. Given previous reports of the influence of cholesterol on other receptor tyrosine kinases (RTKs), as well as the modulation of lipid composition in cancer cells, we wished to investigate how cholesterol content impacts the structure of the Neu transmembrane domain. We utilized high-resolution magic angle spinning solid-state NMR to measure 13C-13C coupling of selectively labelled probe residues in the Neu transmembrane domain in lipid bilayers containing cholesterol. We observe inter-helical coupling between residues that support helix-helix interactions on both dimerization motifs reported in the literature (A661-XXX-G665 and I659-XXX-V663). We further explore how changes in cholesterol concentration alter transmembrane domain interactions and the properties and mechanics of the bilayer. We interpret our results in light of previous studies relating RTK activity to cholesterol enrichment and/or depletion, and propose a novel model to explain our data that includes the recognition and binding of cholesterol by the Neu transmembrane domain through a putative cholesterol-recognition/interaction amino acid consensus sequence.

Huntingtin exon 1 fibrils feature an interdigitated ß-hairpin-based polyglutamine core.

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of ß-hairpin-containing ß-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical ß-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

Protein-protein interactions in trans-AT polyketide synthases.

Covering: up to 2018 The construction of polyketide natural products by type I modular polyketide synthases (PKSs) requires the coordinated action of several protein subunits to ensure biosynthetic fidelity. This is particularly the case for trans-AT PKSs, which in contrast to most cis-AT PKSs, contain split modules and employ several trans-acting catalytic domains. This article summarises recent advances in understanding the protein-protein interactions underpinning subunit assembly and intra-subunit communication in such systems and highlights potential avenues and approaches for future research.

Solid-State NMR Provides Evidence for Small-Amplitude Slow Domain Motions in a Multispanning Transmembrane α-Helical Protein.

Proteins are dynamic entities and populate ensembles of conformations. Transitions between states within a conformational ensemble occur over a broad spectrum of amplitude and time scales, and are often related to biological function. Whereas solid-state NMR (SSNMR) spectroscopy has recently been used to characterize conformational ensembles of proteins in the microcrystalline states, its applications to membrane proteins remain limited. Here we use SSNMR to study conformational dynamics of a seven-helical transmembrane (TM) protein, Anabaena Sensory Rhodopsin (ASR) reconstituted in lipids. We report on site-specific measurements of the 15N longitudinal R1 and rotating frame R1ρ relaxation rates at two fields of 600 and 800 MHz and at two temperatures of 7 and 30 °C. Quantitative analysis of the R1 and R1ρ values and of their field and temperature dependencies provides evidence of motions on at least two time scales. We modeled these motions as fast local motions and slower collective motions of TM helices and of structured loops, and used the simple model-free and extended model-free analyses to fit the data and estimate the amplitudes, time scales and activation energies. Faster picosecond (tens to hundreds of picoseconds) local motions occur throughout the protein and are dominant in the middle portions of the TM helices. In contrast, the amplitudes of the slower collective motions occurring on the nanosecond (tens to hundreds of nanoseconds) time scales, are smaller in the central parts of helices, but increase toward their cytoplasmic sides as well as in the interhelical loops. ASR interacts with a soluble transducer protein on its cytoplasmic surface, and its binding affinity is modulated by light. The larger amplitude of motions on the cytoplasmic side of the TM helices correlates with the ability of ASR to undergo large conformational changes in the process of binding/unbinding the transducer.

Characterization of Protein-Protein Interfaces in Large Complexes by Solid-State NMR Solvent Paramagnetic Relaxation Enhancements.

Solid-state NMR is becoming a viable alternative for obtaining information about structures and dynamics of large biomolecular complexes, including ones that are not accessible to other high-resolution biophysical techniques. In this context, methods for probing protein-protein interfaces at atomic resolution are highly desirable. Solvent paramagnetic relaxation enhancements (sPREs) proved to be a powerful method for probing protein-protein interfaces in large complexes in solution but have not been employed toward this goal in the solid state. We demonstrate that 1H and 15N relaxation-based sPREs provide a powerful tool for characterizing intermolecular interactions in large assemblies in the solid state. We present approaches for measuring sPREs in practically the entire range of magic angle spinning frequencies used for biomolecular studies and discuss their benefits and limitations. We validate the approach on crystalline GB1, with our experimental results in good agreement with theoretical predictions. Finally, we use sPREs to characterize protein-protein interfaces in the GB1 complex with immunoglobulin G (IgG). Our results suggest the potential existence of an additional binding site and provide new insights into GB1:IgG complex structure that amend and revise the current model available from studies with IgG fragments. We demonstrate sPREs as a practical, widely applicable, robust, and very sensitive technique for determining intermolecular interaction interfaces in large biomolecular complexes in the solid state.

Unraveling the complexity of protein backbone dynamics with combined (13)C and (15)N solid-state NMR relaxation measurements.

Typically, protein dynamics involve a complex hierarchy of motions occurring on different time scales between conformations separated by a range of different energy barriers. NMR relaxation can in principle provide a site-specific picture of both the time scales and amplitudes of these motions, but independent relaxation rates sensitive to fluctuations in different time scale ranges are required to obtain a faithful representation of the underlying dynamic complexity. This is especially pertinent for relaxation measurements in the solid state, which report on dynamics in a broader window of time scales by more than 3 orders of magnitudes compared to solution NMR relaxation. To aid in unraveling the intricacies of biomolecular dynamics we introduce (13)C spin-lattice relaxation in the rotating frame (R1ρ) as a probe of backbone nanosecond-microsecond motions in proteins in the solid state. We present measurements of (13)C'R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz (1)H Larmor frequencies and compare them to (13)C'R1, (15)N R1 and R1ρ measured under the same conditions. The addition of carbon relaxation data to the model free analysis of nitrogen relaxation data leads to greatly improved characterization of time scales of protein backbone motions, minimizing the occurrence of fitting artifacts that may be present when (15)N data is used alone. We also discuss how internal motions characterized by different time scales contribute to (15)N and (13)C relaxation rates in the solid state and solution state, leading to fundamental differences between them, as well as phenomena such as underestimation of picosecond-range motions in the solid state and nanosecond-range motions in solution.

Intermolecular Interactions and Protein Dynamics by Solid-State NMR Spectroscopy.

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1-antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site-specific (15) N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond-nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small-amplitude overall anisotropic motion sampling the interaction interface in the complex.

Solid-state NMR of a protein in a precipitated complex with a full-length antibody.

NMR spectroscopy is a prime technique for characterizing atomic-resolution structures and dynamics of biomolecular complexes but for such systems faces challenges of sensitivity and spectral resolution. We demonstrate that the application of (1)H-detected experiments at magic-angle spinning frequencies of >50 kHz enables the recording, in a matter of minutes to hours, of solid-state NMR spectra suitable for quantitative analysis of protein complexes present in quantities as small as a few nanomoles (tens of micrograms for the observed component). This approach enables direct structure determination and quantitative dynamics measurements in domains of protein complexes with masses of hundreds of kilodaltons. Protein-protein interaction interfaces can be mapped out by comparison of the chemical shifts of proteins within solid-state complexes with those of the same constituent proteins free in solution. We employed this methodology to characterize a >300 kDa complex of GB1 with full-length human immunoglobulin, where we found that sample preparation by simple precipitation yields spectra of exceptional quality, a feature that is likely to be shared with some other precipitating complexes. Finally, we investigated extensions of our methodology to spinning frequencies of up to 100 kHz.

Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR.

The ability to detect and characterize molecular motions represents one of the unique strengths of nuclear magnetic resonance (NMR) spectroscopy. In this study, we report solid-state NMR site-specific measurements of the dipolar order parameters and (15)N rotating frame spin-lattice (R1ρ) relaxation rates in a seven transmembrane helical protein Anabaena Sensory Rhodopsin reconstituted in lipids. The magnitudes of the observed order parameters indicate that both the well-defined transmembrane regions and the less structured intramembrane loops undergo restricted submicrosecond time scale motions. In contrast, the R1ρ rates, which were measured under fast magic angle spinning conditions, vary by an order of magnitude between the TM and exposed regions and suggest the presence of intermediate time scale motions. Using a simple model, which assumes a single exponential autocorrelation function, we estimated the time scales of dominant stochastic motions to be on the order of low tens of nanoseconds for most residues within the TM helices and tens to hundreds of nanoseconds for the extracellular B-C and F-G loops. These relatively slow time scales could be attributed to collective anisotropic motions. We used the 3D Gaussian axial fluctuations model to estimate amplitudes, directions, and time scales of overall motions for helices and the extracellular B-C and F-G loops. Within this model, the TM helices A,B,C,D,E,F undergo rigid body motions on a time scale of tens of nanoseconds, while the time scale for the seventh helix G approaches 100 ns. Similar time scales of roughly 100-200 ns are estimated for the B-C and F-G loops.

Molecular basis for short-chain thioester hydrolysis by acyl hydrolase domains in trans -acyltransferase polyketide synthases.

Polyketide synthases (PKSs) are multi-domain enzymatic assembly lines that biosynthesise a wide selection of bioactive natural products from simple building blocks. In contrast to their cis -acyltransferase (AT) counterparts, trans -AT PKSs rely on stand-alone AT domains to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery. Trans -AT PKS gene clusters also encode acyl hydrolase (AH) domains, which are predicted to share the overall fold of AT domains, but hydrolyse aberrant acyl chains from ACP domains, thus ensuring efficient polyketide biosynthesis. How such domains specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH domain and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AH and ACP domains contributes to recognition of cognate and non-cognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the production of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis by trans -AT PKSs, and provide foundations for future bioengineering efforts.

Compensated second-order recoupling: application to third spin assisted recoupling.

We consider the effect of phase shifts in the context of second-order recoupling techniques in solid-state NMR. Notably we highlight conditions leading to significant improvements for the Third Spin Assisted Recoupling (TSAR) mechanism and demonstrate the benefits of resulting techniques for detecting long-distance transfer in biomolecular systems. The modified pulse sequences of PAR and PAIN-CP, Phase-Shifted Proton Assisted Recoupling (AH-PS-PAR) and Phase-Shifted Proton-Assisted Insensitive Nuclei Cross Polarization (ABH-PS-PAIN-CP), still rely on cross terms between heteronuclear dipolar couplings involving assisting protons that mediate zero-quantum polarization transfer between low-γ nuclei ((13)C-(13)C, (15)N-(15)N, (15)N-(13)C polarization transfer). Using Average Hamiltonian Theory we show that phase inversion compensates off-resonance contributions and yields improved polarization transfer as well as substantial broadening of the matching conditions. PS-TSAR greatly improves on the standard TSAR based methods because it alleviates their sensitivity to precise RF settings which significantly enhances robustness of the experiments. We demonstrate these new methods on a 19.6 kDa protein (U-[(15)N, (13)C]-YajG) at high magnetic fields (up to 900 MHz (1)H frequency) and fast sample spinning (up to 65 kHz MAS frequency).

Site-specific measurement of slow motions in proteins.

We demonstrate that a quantitative measure of slow molecular motions in solid proteins can be accessed by measuring site-specific (15)N rotating-frame relaxation rates at high magic-angle-spinning frequencies.