From Milliseconds to Minutes: Melittin Self-Assembly from Concerted Non-Equilibrium Pressure-Jump and Equilibrium Relaxation Nuclear Magnetic Resonance.

Non-equilibrium kinetics techniques like pressure-jump nuclear magnetic resonance (NMR) are powerful in tracking changes in oligomeric populations and are not limited by relaxation rates for the time scales of exchange that can be probed. However, these techniques are less sensitive to minor, transient populations than are Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. We integrated non-equilibrium pressure-jump and equilibrium CPMG relaxation dispersion data to fully map the kinetic landscape of melittin tetramerization. While monomeric peptides weakly form dimers (Kd,D/M ≈ 26 mM) whose population never exceeds 1.6% at 288 K, dimers associate tightly to form stable tetrameric species (Kd,T/D ≈ 740 nM). Exchange between the monomer and dimer, along with exchange between the dimer and tetramer, occurs on the millisecond time scale. The NMR approach developed herein can be readily applied to studying the folding and misfolding of a wide range of oligomeric assemblies.

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in E. coli. Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and is thus a prerequisite for studying its native structure and function. Here, we present a method for producing triply isotopically labeled (2H, 13C, and 15N) native melittin through recombinant expression followed by chemical amidation. We then show that structural models produced with AlphaFold-Multimer are in even better agreement with experimental residual dipolar couplings than the 2.0 Å resolution X-ray crystal structure for residues G3-K23.

From Angstroms to Nanometers: Measuring Interatomic Distances by Solid-State NMR.

Internuclear distances represent one of the main structural constraints in molecular structure determination using solid-state NMR spectroscopy, complementing chemical shifts and orientational restraints. Although a large number of magic-angle-spinning (MAS) NMR techniques have been available for distance measurements, traditional 13C and 15N NMR experiments are inherently limited to distances of a few angstroms due to the low gyromagnetic ratios of these nuclei. Recent development of fast MAS triple-resonance 19F and 1H NMR probes has stimulated the design of MAS NMR experiments that measure distances in the 1-2 nm range with high sensitivity. This review describes the principles and applications of these multiplexed multidimensional correlation distance NMR experiments, with an emphasis on 19F- and 1H-based distance experiments. Representative applications of these long-distance NMR methods to biological macromolecules as well as small molecules are reviewed.

Off-resonance 13C-2H REDOR NMR for site-resolved studies of molecular motion.

We introduce a 13C-2H Rotational Echo DOuble Resonance (REDOR) technique that uses the difference between on-resonance and off-resonance 2H irradiation to detect dynamic segments in deuterated molecules. By selectively inverting specific regions of the 2H magic-angle spinning (MAS) sideband manifold to recouple some of the deuterons to nearby carbons, we distinguish dynamic and rigid residues in 1D and 2D 13C spectra. We demonstrate this approach on deuterated GB1, H/D exchanged GB1, and perdeuterated bacterial cellulose. Numerical simulations reproduce the measured mixing-time and 2H carrier-frequency dependence of the REDOR dephasing of bacterial cellulose. Combining numerical simulations with experiments thus allow the extraction of motionally averaged quadrupolar couplings from REDOR dephasing values.

Structurally Based Design of Glucagon Mutants That Inhibit Fibril Formation.

The peptide hormone glucagon is prescribed as a pharmaceutical compound to treat diabetic hypoglycemia. However, at the acidic pH where it is highly soluble, glucagon rapidly aggregates into inactive and cytotoxic amyloid fibrils. The recently determined high-resolution structure of these fibrils revealed various stabilizing molecular interactions. On the basis of this structure, we have now designed four arginine mutants of glucagon that resist fibrillization at pharmaceutical concentrations for weeks. An S2R, T29R double mutant and a T29R single mutant remove a hydrogen-bonding interaction in the wild-type fibril, whereas a Y13R, A19R double mutant and a Y13R mutant remove a cation-π interaction. 1H solution nuclear magnetic resonance spectra and ultraviolet absorbance data indicate that these mutants remain soluble in pH 2 buffer under quiescent conditions at concentrations of ≤4 mg/mL for weeks. Under stressed conditions with high salt concentrations and agitation, these mutants fibrillize significantly more slowly than the wild type. The S2R, T29R mutant and the T29R mutant exhibit a mixture of random coil and α-helical conformations, while the Y13R mutant is completely random coil. The mutation sites are chosen to be uninvolved in strong interactions with the glucagon receptor in the active structure of the peptide. Therefore, these arginine mutants of glucagon are promising alternative compounds for treating hypoglycemia.

Comparative analysis of 13C chemical shifts of ß-sheet amyloid proteins and outer membrane proteins.

Cross-ß amyloid fibrils and membrane-bound ß-barrels are two important classes of ß-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the 13C chemical shifts of 17 amyloid proteins and 7 ß-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 ß-sheet residues in amyloid fibrils and 521 ß-sheet residues in ß-barrel membrane proteins. The 13C chemical shifts are shown in 2D 13C-13C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in ß-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and ß-barrels and compare the sidechain rotameric populations. The 13C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in ß-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in ß-barrels. These trends can be explained by steric zipper interactions between ß-sheet planes in cross-ß fibrils, and by the interactions of ß-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and ß-barrels based principally on NMR chemical shifts.

Water orientation and dynamics in the closed and open influenza B virus M2 proton channels.

The influenza B M2 protein forms a water-filled tetrameric channel to conduct protons across the lipid membrane. To understand how channel water mediates proton transport, we have investigated the water orientation and dynamics using solid-state NMR spectroscopy and molecular dynamics (MD) simulations. 13C-detected water 1H NMR relaxation times indicate that water has faster rotational motion in the low-pH open channel than in the high-pH closed channel. Despite this faster dynamics, the open-channel water shows higher orientational order, as manifested by larger motionally-averaged 1H chemical shift anisotropies. MD simulations indicate that this order is induced by the cationic proton-selective histidine at low pH. Furthermore, the water network has fewer hydrogen-bonding bottlenecks in the open state than in the closed state. Thus, faster dynamics and higher orientational order of water molecules in the open channel establish the water network structure that is necessary for proton hopping.

Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range 13C-13C and 15N-13C Distance Restraints.

We present a class of pulsed third-spin-assisted recoupling (P-TSAR) magic-angle-spinning solid-state NMR techniques that achieve efficient polarization transfer over long distances to provide important restraints for structure determination. These experiments utilize second-order cross terms between strong 1H-13C and 1H-15N dipolar couplings to achieve 13C-13C and 15N-13C polarization transfer, similar to the principle of continuous-wave (CW) TSAR experiments. However, in contrast to the CW-TSAR experiments, these P-TSAR experiments require much less radiofrequency (rf) energy and allow a much simpler routine for optimizing the rf field strength. We call the technique PULSAR (pulsed proton-assisted recoupling) for homonuclear spin pairs. For heteronuclear spin pairs, we improve the recently introduced PERSPIRATIONCP (proton-enhanced rotor-echo short pulse irradiation cross-polarization) experiment by shifting the pulse positions and removing the z-filters, which significantly broaden the bandwidth and increase the efficiency of polarization transfer. We demonstrate the PULSAR and PERSPIRATIONCP techniques on the model protein GB1 and found cross peaks for distances as long as 10 and 8 Å for 13C-13C and 15N-13C spin pairs, respectively. We then apply these methods to the amyloid fibrils formed by the peptide hormone glucagon and show that long-range correlation peaks are readily observed to constrain intermolecular packing in this cross-ß fibril. We provide an analytical model for the PULSAR and PERSPIRATIONCP experiments to explain the measured and simulated chemical shift dependence and pulse flip angle dependence of polarization transfer. These two techniques are useful for measuring long-range distance restraints to determine the three-dimensional structures of proteins and other biological macromolecules.

The peptide hormone glucagon forms amyloid fibrils with two coexisting ß-strand conformations.

Glucagon and insulin maintain blood glucose homeostasis and are used to treat hypoglycemia and hyperglycemia, respectively, in patients with diabetes. Whereas insulin is stable for weeks in its solution formulation, glucagon fibrillizes rapidly at the acidic pH required for solubility and is therefore formulated as a lyophilized powder that is reconstituted in an acidic solution immediately before use. Here we use solid-state NMR to determine the atomic-resolution structure of fibrils of synthetic human glucagon grown at pharmaceutically relevant low pH. Unexpectedly, two sets of chemical shifts are observed, indicating the coexistence of two ß-strand conformations. The two conformations have distinct water accessibilities and intermolecular contacts, indicating that they alternate and hydrogen bond in an antiparallel fashion along the fibril axis. Two antiparallel ß-sheets assemble with symmetric homodimer cross sections. This amyloid structure is stabilized by numerous aromatic, cation-π, polar and hydrophobic interactions, suggesting mutagenesis approaches to inhibit fibrillization could improve this important drug.

The Transmembrane Conformation of the Influenza B Virus M2 Protein in Lipid Bilayers.

Influenza A and B viruses cause seasonal flu epidemics. The M2 protein of influenza B (BM2) is a membrane-embedded tetrameric proton channel that is essential for the viral lifecycle. BM2 is a functional analog of AM2 but shares only 24% sequence identity for the transmembrane (TM) domain. The structure and function of AM2, which is targeted by two antiviral drugs, have been well characterized. In comparison, much less is known about the structure of BM2 and no drug is so far available to inhibit this protein. Here we use solid-state NMR spectroscopy to investigate the conformation of BM2(1-51) in phospholipid bilayers at high pH, which corresponds to the closed state of the channel. Using 2D and 3D correlation NMR experiments, we resolved and assigned the 13C and 15N chemical shifts of 29 residues of the TM domain, which yielded backbone (φ, ψ) torsion angles. Residues 6-28 form a well-ordered α-helix, whereas residues 1-5 and 29-35 display chemical shifts that are indicative of random coil or ß-sheet conformations. The length of the BM2-TM helix resembles that of AM2-TM, despite their markedly different amino acid sequences. In comparison, large 15N chemical shift differences are observed between bilayer-bound BM2 and micelle-bound BM2, indicating that the TM helix conformation and the backbone hydrogen bonding in lipid bilayers differ from the micelle-bound conformation. Moreover, HN chemical shifts of micelle-bound BM2 lack the periodic trend expected for coiled coil helices, which disagree with the presence of a coiled coil structure in micelles. These results establish the basis for determining the full three-dimensional structure of the tetrameric BM2 to elucidate its proton-conduction mechanism.

Efficient 15N-13C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for Protein Structure Determination.

We introduce a pulsed third-spin-assisted recoupling experiment that produces high-intensity long-range 15N-13C cross peaks using low radiofrequency (rf) energy. This Proton-Enhanced Rotor-echo Short-Pulse IRradiATION Cross-Polarization (PERSPIRATIONCP) pulse sequence operates with the same principle as the Proton-Assisted Insensitive-Nuclei Cross-Polarization (PAINCP) experiment but uses only a fraction of the rf energy by replacing continuous-wave 13C and 15N irradiation with rotor-echo 90° pulses. Using formyl-Met-Leu-Phe (f-MLF) and ß1 immunoglobulin binding domain of protein G (GB1) as model proteins, we demonstrate experimentally how PERSPIRATIONCP polarization transfer depends on the CP contact time, rf power, pulse flip angle, and 13C carrier frequency and compare the PERSPIRATIONCP performance with the performances of PAINCP, RESPIRATIONCP, and SPECIFICCP for measuring 15N-13C cross peaks. PERSPIRATIONCP achieves long-range 15N-13C transfer and yields higher cross peak-intensities than that of the other techniques. Numerical simulations reproduce the experimental trends and moreover indicate that PERSPIRATIONCP relies on 15N-1H and 13C-1H dipolar couplings rather than 15N-13C dipolar coupling for polarization transfer. Therefore, PERSPIRATIONCP is an rf-efficient and higher-sensitivity alternative to PAINCP for measuring long-range 15N-13C correlations, which are essential for protein resonance assignment and structure determination. Using cross peaks from two PERSPIRATIONCP 15N-13C correlation spectra as the sole distance restraints, supplemented with (φ, ψ) torsion angles obtained from chemical shifts, we calculated the GB1 structure and obtained a backbone root-mean-square deviation of 2.0 Å from the high-resolution structure of the protein. Therefore, this rf-efficient PERSPIRATIONCP method is useful for obtaining many long-range distance restraints for protein structure determination.

Transport-Relevant Protein Conformational Dynamics and Water Dynamics on Multiple Time Scales in an Archetypal Proton Channel: Insights from Solid-State NMR.

The influenza M2 protein forms a tetrameric proton channel that conducts protons from the acidic endosome into the virion by shuttling protons between water and a transmembrane histidine. Previous NMR studies have shown that this histidine protonates and deprotonates on the microsecond time scale. However, M2's proton conduction rate is 10-1000 s-1, more than 2 orders of magnitude slower than the histidine-water proton-exchange rate. M2 is also known to be conformationally plastic. To address the disparity between the functional time scale and the time scales of protein conformational dynamics and water dynamics, we have now investigated a W41F mutant of the M2 transmembrane domain using solid-state NMR. 13C chemical shifts of the membrane-bound peptide indicate the presence of two distinct tetramer conformations, whose concentrations depend exclusively on pH and hence the charge-state distribution of the tetramers. High-temperature 2D correlation spectra indicate that these two conformations interconvert at a rate of ∼400 s-1 when the +2 and +3 charge states dominate, which gives the first experimental evidence of protein conformational motion on the transport time scale. Protein 13C-detected water 1H T2 relaxation measurements show that channel water relaxes an order of magnitude faster than bulk water and membrane-associated water, indicating that channel water undergoes nanosecond motion in a pH-independent fashion. These results connect motions on three time scales to explain M2's proton-conduction mechanism: picosecond-to-nanosecond motions of water molecules facilitate proton Grotthuss hopping, microsecond motions of the histidine side chain allow water-histidine proton transfer, while millisecond motions of the entire four-helix bundle constitute the rate-limiting step, dictating the number of protons released into the virion.

Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR.

The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon-fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid 31P spectra show an isotropic peak indicative of high membrane curvature. This M2-cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol's effects on membrane protein function.

2H-13C correlation solid-state NMR for investigating dynamics and water accessibilities of proteins and carbohydrates.

Site-specific determination of molecular motion and water accessibility by indirect detection of 2H NMR spectra has advantages over dipolar-coupling based techniques due to the large quadrupolar couplings and the ensuing high angular resolution. Recently, a Rotor Echo Short Pulse IRrAdiaTION mediated cross polarization (RESPIRATIONCP) technique was developed, which allowed efficient transfer of 2H magnetization to 13C at moderate 2H radiofrequency field strengths available on most commercial MAS probes. In this work, we investigate the 2H-13C magnetization transfer characteristics of one-bond perdeuterated CD n spin systems and two-bond H/D exchanged C-(O)-D and C-(N)-D spin systems in carbohydrates and proteins. Our results show that multi-bond, broadband 2H-13C polarization transfer can be achieved using 2H radiofrequency fields of ~50 kHz, relatively short contact times of 1.3-1.7 ms, and with sufficiently high sensitivity to enable 2D 2H-13C correlation experiments with undistorted 2H spectra in the indirect dimension. To demonstrate the utility of this 2H-13C technique for studying molecular motion, we show 2H-13C correlation spectra of perdeuterated bacterial cellulose, whose surface glucan chains exhibit a motionally averaged C6 2H quadrupolar coupling that indicates fast trans-gauche isomerization about the C5-C6 bond. In comparison, the interior chains in the microfibril core are fully immobilized. Application of the 2H-13C correlation experiment to H/D exchanged Arabidopsis primary cell walls show that the O-D quadrupolar spectra of the highest polysaccharide peaks can be fit to a two-component model, in which 74% of the spectral intensity, assigned to cellulose, has a near-rigid-limit coupling, while 26% of the intensity, assigned to matrix polysaccharides, has a weakened coupling of 50 kHz. The latter O-D quadrupolar order parameter of 0.22 is significantly smaller than previously reported C-D dipolar order parameters of 0.46-0.55 for pectins, suggesting that additional motions exist at the C-O bonds in the wall polysaccharides. 2H-13C polarization transfer profiles are also compared between statistically deuterated and H/D exchanged GB1.

Icephobic Surfaces Induced by Interfacial Nonfrozen Water.

It is known that smooth, hydrophobic solid surfaces exhibit low ice adhesion values, which have been shown to approach a lower ice adhesion strength limit (∼150 kPa) defined by the water receding contact angle. To overcome this limit, we have designed self-lubricating icephobic coatings by blending polydimethylsiloxane (PDMS)-poly(ethylene glycol) (PEG) amphiphilic copolymers into a polymer matrix. Such coatings provide low ice adhesion strength values (∼50 kPa) that can substantially reduce the lower bound of the ice adhesion strength achieved previously on smooth, hydrophobic solid surfaces. Different molecular mechanisms are responsible for the low ice adhesion strength attained by these two approaches. For the smooth hydrophobic surfaces, an increased water depletion layer thickness at the interface weakens the van der Waals' interactions between the ice and the polymeric substrate. For the self-lubricating icephobic coatings, the PEG component of the amphiphilic copolymer is capable of strongly hydrogen bonding with water molecules. The surface hydrogen-bonded water molecules do not freeze, even at substantial levels of subcooling, and therefore serve as a self-lubricating interfacial liquid-like layer that helps to reduce the adhesion strength of ice to the surface. The existence of nonfrozen water molecules at the ice-solid interface is confirmed by solid-state nuclear magnetic resonance (NMR) spectroscopy.