An Off‐Pathway Folding Intermediate of an Acyl Carrier Protein Domain Coexists with the Folded and Unfolded States under Native Conditions

A protein can exist in multiple states under native conditions and those states with low populations are often critical to biological function and self‐assembly. To investigate the role of the minor states of an acyl carrier protein, NMR techniques were applied to determine the number of minor states and characterize their structures and kinetics. The acyl carrier protein from Micromonospora echinospora was found to exist in one major folded state (95.2 %), one unfolded state (4.1 %), and one intermediate state (0.7 %) under native conditions. The three states are in dynamic equilibrium and the intermediate state very likely adopts a native‐like structure and is an off‐pathway folding product. The intermediate state may mediate the formation of oligomers in vitro and play an important role in the recognition of partner enzymes in vivo.     

Conformational Dynamics of apo‐GlnBP Revealed by Experimental and Computational Analysis

The glutamine binding protein (GlnBP) binds l ‐glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo‐ and holo‐GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single‐molecule FRET techniques to decipher the conformational dynamics of apo‐GlnBP. The NMR residual dipolar couplings of apo‐GlnBP were in good agreement with a MD‐derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four‐state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP.     

Conformational Plasticity in Glycomimetics: Fluorocarbamethyl‐L‐idopyranosides Mimic the Intrinsic Dynamic Behaviour of Natural Idose Rings

Sugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsically related to biological activity; actually plasticity in L ‐iduronic rings modulates their interactions with biological receptors. However, the access to the experimental values of the energy barriers and free‐energy difference for conformer interconversion in water solution has been elusive. Here, a new generation of fluorine‐containing glycomimetics is presented. We have applied a combination of organic synthesis, NMR spectroscopy and computational methods to investigate the conformational behaviour of idose‐ and glucose‐like rings. We have used low‐temperature NMR spectroscopic experiments to slow down the conformational exchange of the idose‐like rings. Under these conditions, the exchange rate becomes slow in the ¹⁹F NMR spectroscopic chemical shift timescale and allows shedding light on the thermodynamic and kinetic features of the equilibrium. Despite the minimal structural differences between these compounds, a remarkable difference in their dynamic behaviour indeed occurs. The importance of introducing fluorine atoms in these sugars mimics is also highlighted. Only the use of ¹⁹F NMR spectroscopic experiments has permitted the unveiling of key features of the conformational equilibrium that would have otherwise remained unobserved.     

A Methyl‐TROSY‐Based 1H Relaxation Dispersion Experiment for Studies of Conformational Exchange in High Molecular Weight Proteins

Molecular complexes often sample conformational states that direct them to specific functions. These states can be difficult to observe through traditional biophysical approaches but they can be studied using a variety of different NMR spin relaxation experiments. However, these applications, when focused on moderate to high molecular weight proteins, are complicated by fast relaxing signals that negatively affect the sensitivity and resolution of spectra. Here a methyl ¹H CPMG‐based experiment for studies of excited conformational states of protein machines is described that exploits a TROSY‐effect to increase signal‐to‐noise. Complexities from the multiplicity of methyl ¹H transitions are addressed to generate a robust pulse scheme that is applied to a 320 kDa homeostasis protein, p97.     

Functional Dynamics of Deuterated β2‐Adrenergic Receptor in Lipid Bilayers Revealed by NMR Spectroscopy

G‐protein‐coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β₂‐adrenergic receptor (β₂AR) in lipid bilayers of reconstituted high‐density lipoprotein (rHDL) increased by approximately 5‐fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β₂AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception.     

Probing Transient Conformational States of Proteins by Solid‐State R1ρ Relaxation‐Dispersion NMR Spectroscopy

The function of proteins depends on their ability to sample a variety of states differing in structure and free energy. Deciphering how the various thermally accessible conformations are connected, and understanding their structures and relative energies is crucial in rationalizing protein function. Many biomolecular reactions take place within microseconds to milliseconds, and this timescale is therefore of central functional importance. Here we show that R_1ρ relaxation dispersion experiments in magic‐angle‐spinning solid‐state NMR spectroscopy make it possible to investigate the thermodynamics and kinetics of such exchange process, and gain insight into structural features of short‐lived states.     

Dynamic NMR study of dinitrophenyl derivatives of seven‐membered cyclic ketals of pyridoxine

Two pyridoxine derivatives containing a dinitrophenyl moiety were investigated by ¹H NMR spectroscopy. Conformational dynamics in solution were studied for each compound using dynamic NMR experiments. It was shown that both compounds studied are involved into two conformational exchange processes. The first process is a transformation of the seven‐membered cycle conformation between the enantiomeric P‐twist and M‐twist forms, and the second is a rotation of the dinitrophenyl fragment of the molecules around the C–O bond. Energy barriers of both conformational transitions were determined. Copyright © 2015 John Wiley & Sons, Ltd.     

Allosteric conformational changes of G proteins upon its interaction with membrane and GPCR

Current resolved structures of GPCRs and G protein complexes provided important insights into G protein activation. However, the binding or dissociation of GPCRs with G protein is instantaneous and highly dynamic in the intracellular environment. The conformational dynamic of G protein still needs to be addressed. In this study, we applied19 F solution NMR spectroscopy to monitor the conformational changes of G protein upon interact with detergent mimicking membrane and receptor. Our results sho...     

Assessment of a Large Enzyme–Drug Complex by Proton‐Detected Solid‐State NMR Spectroscopy without Deuteration

Solid‐state NMR spectroscopy has recently enabled structural biology with small amounts of non‐deuterated proteins, largely alleviating the classical sample production demands. Still, despite the benefits for sample preparation, successful and comprehensive characterization of complex spin systems in the few cases of higher‐molecular‐weight proteins has thus far relied on traditional ¹³C‐detected methodology or sample deuteration. Herein we show for a 29 kDa carbonic anhydrase:acetazolamide complex that different aspects of solid‐state NMR assessment of a complex spin system can be successfully accessed using a non‐deuterated, 500 μg sample in combination with adequate spectroscopic tools. The shown access to protein structure, protein dynamics, as well as biochemical parameters in amino acid sidechains, such as histidine protonation states, will be transferable to proteins that are not expressible in E. coli.     

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by 13C solid‐state and 1H time‐domain NMR

Monitoring of the molecular motions and secondary structures of gliadin (Gli) and glutenin (Glu) in blends with 10, 20, 30, and 40% glycerol was performed by solid‐state (SS) and time domain (TD) NMR spectroscopy. Increasing the glycerol content increased the relative amount of β‐sheets and disordered structures, while decreasing α‐helices in Gli/Glu–glycerol blends studied by ¹³C CPMAS NMR. For ≥20% glycerol samples, the protein side‐chain mobility increased similarly for Gli and Glu. A higher proportion of α‐helices versus β‐sheets was found in Gli‐glycerol blends compared with Glu–glycerol blends. Glycerol acted as “immobilized” in 10–20% glycerol Gli samples and was found mainly “free” in 30 and 40% glycerol Gli/Glu samples. During temperature experiments, 30 and 40% glycerol amounts impacted the dynamic molecular behavior of the Gli and Glu proteins differently than lipids, as observed by TD‐NMR. The combination of TD‐NMR together with SS‐NMR showed details of the dynamic molecular variations in Gli/Glu protein structure and are promising techniques to monitor the molecular dynamics of plasticized proteins. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 739–750     

Enhancing the Sensitivity of CPMG Relaxation Dispersion to Conformational Exchange Processes by Multiple‐Quantum Spectroscopy

A triple‐quantum ¹H Carr–Purcell–Meiboom–Gill NMR relaxation dispersion experiment is presented that uses methyl group probes as reporters of conformational exchange in highly deuterated, methyl‐protonated proteins. Significantly larger dispersion profiles, by as much as a factor of nine, can be obtained relative to single‐quantum approaches, thus offering very significant advantages in applications involving interconverting conformers with only small changes in structure or in studies of rare states that are at very low populations. Applications to a number of protein systems are presented where the utility of the method, including its improved sensitivity to chemical exchange processes, is established.     

Conformational Changes in Calcium‐Sensor Proteins under Molecular Crowding Conditions

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca²⁺ sensors, which undergo conformational changes in response to oscillating intracellular Ca²⁺ concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca²⁺‐binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase‐activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca²⁺‐sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca²⁺ conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size‐exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein–water interface. Our study provides insight into how rather small signaling proteins that have very similar three‐dimensional folding patterns differ in their Ca²⁺‐occupied functional state under crowding conditions.     

Microsecond Timescale Dynamics of GDP‐Bound Ras Underlies the Formation of Novel Inhibitor‐Binding Pockets

The recent discovery of inhibitory compounds binding to distinct pockets on GDP‐bound Ras has renewed the view on the druggability of this crucial cancer driver. However, the origin of these pockets, which are not readily formed in the crystal structure in the absence of the compounds, is yet unclear. Herein, we explored the intrinsic flexibility of Ras?GDP on microsecond to millisecond timescales using relaxation‐based NMR experiments, and identified substantial slow dynamics with τ_ex of 34 μs at 5 °C, which maps to the regions showing a high level of correlation with those displaying conformational differences between the inhibitor‐bound and free states. These findings, which have been demonstrated in both wild‐type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras–inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.     

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

Site‐Specific Solid‐State NMR Studies of “Trigger Factor” in Complex with the Large Ribosomal Subunit 50S

Co‐translational protein folding is not yet well understood despite the availability of high‐resolution ribosome crystal structures. We present first solid‐state NMR data on non‐mobile regions of a prokaryotic ribosomal complex. Localized chemical shift perturbations and line broadening are observed for the backbone amide resonances corresponding to the regions in the trigger factor ribosome‐binding domain that are involved in direct contact with the ribosome or undergo conformational changes upon ribosome binding. This large asymmetric protein complex (1.4 MDa) becomes accessible for NMR investigations by the combined use of proton detection and high MAS frequencies (60 kHz). The presented results open new perspectives for the understanding of the mechanism of large molecular machineries.     

Characterizing Methyl‐Bearing Side Chain Contacts and Dynamics Mediating Amyloid β Protofibril Interactions Using 13Cmethyl‐DEST and Lifetime Line Broadening

Many details pertaining to the formation and interactions of protein aggregates associated with neurodegenerative diseases are invisible to conventional biophysical techniques. We recently introduced ¹⁵N dark‐state exchange saturation transfer (DEST) and ¹⁵N lifetime line‐broadening to study solution backbone dynamics and position‐specific binding probabilities for amyloid β (Aβ) monomers in exchange with large (2–80 MDa) protofibrillar Aβ aggregates. Here we use ¹³C_methyl DEST and lifetime line‐broadening to probe the interactions and dynamics of methyl‐bearing side chains in the Aβ‐protofibril‐bound state. We show that all methyl groups of Aβ40 populate direct‐contact bound states with a very fast effective transverse relaxation rate, indicative of side‐chain‐mediated direct binding to the protofibril surface. The data are consistent with position‐specific enhancements of ¹³C_methyl‐${R{{{\rm tethered}\hfill \atop 2\hfill}}}$ values in tethered states, providing further insights into the structural ensemble of the protofibril‐bound state.     

Heterochromatin Protein HP1α Gelation Dynamics Revealed by Solid‐State NMR Spectroscopy

Heterochromatin protein 1α (HP1α) undergoes liquid–liquid phase separation (LLPS) and forms liquid droplets and gels in vitro, properties that also appear to be central to its biological function in heterochromatin compaction and regulation. Here we use solid‐state NMR spectroscopy to track the conformational dynamics of phosphorylated HP1α during its transformation from the liquid to the gel state. Using experiments designed to probe distinct dynamic modes, we identify regions with varying mobilities within HP1α molecules and show that specific serine residues uniquely contribute to gel formation. The addition of chromatin disturbs the gelation process while preserving the conformational dynamics within individual bulk HP1α molecules. Our study provides a glimpse into the dynamic architecture of dense HP1α phases and showcases the potential of solid‐state NMR to detect an elusive biophysical regime of phase separating biomolecules.     

Far‐ and Mid‐Infrared Spectroscopic Analysis of the Substrate‐Induced Structural Dynamics of Respiratory Complex I

The catalytic activity of the respiratory NADH:ubiquinone oxidoreductase (complex I) is based on conformational reorganizations. Herein we probe the effect of substrates on the conformational flexibility of complex I by means of ¹H/²H exchange kinetics at the level of the amide proton in the mid‐infrared spectral range (1700–1500 cm^?1). Slow, medium, and fast exchanging domains are distinguished that reveal different accessibilities to the solvent. Whereas amide hydrogens undergo rapid exchange with the solvent in an open structure, hydrogens experience much slower exchange when they are involved in H‐bonded structures or when they are sterically inaccessible for the solvent. The results indicate a structure that is more open in the presence of both NADH and quinon. Complementary information on the overall internal hydrogen bonding of the protein was probed in the far infrared (300–30 cm^?1), a spectral range that includes a continuum mode of the hydrogen bonding signature.     

Amide-Exchange-Rate-Edited NMR （AERE-NMR） Experiment： A Novel Method for Resolving Overlapping Resonances

This paper describes an amide-exchange-rate-edited （AERE） NMR method that can effectively alleviate the problem of resonance overlap for proteins and peptides. This method exploits the diversity of amide proton exchange rates and consists of two complementary experiments： （1） SEA （solvent exposed amide）-type NMR experiments to map exchangeable surface residues whose amides are not involved in hydrogen bonding, and （2） presat-type NMR experiments to map solvent inaccessibly buried residues or nonexchangeable residues located in hydrogen-bonded secondary structures with properly controlled saturation transfer via amide proton exchanges with the solvent. This method separates overlapping resonances in a spectrum into two complementary spectra. The AERE-NMR method was demonstrated with a sample of ^15N/^13C/^2H（70%） labeled ribosome-inactivating protein trichosanthin of 247 residues.