Microsecond Motions Research Articles

Substrate inhibition of an enzyme: Are microsecond motions affecting catalytic activity?

Substrate inhibition of an enzyme: Are microsecond motions affecting catalytic activity?

Microsecond Motion of the Bacterial Transporter EmrE in Lipid Bilayers.

The bacterial transporter EmrE is a homo-dimeric membrane protein that effluxes cationic polyaromatic substrates against the concentration gradient by coupling to proton transport. As the archetype of the small multidrug resistance family of transporters, EmrE structure and dynamics provide atomic insights into the mechanism of transport by this family of proteins. We recently determined high-resolution structures of EmrE in complex with a cationic substrate, tetra(4-fluorophenyl)phosphonium (F4-TPP+), using solid-state NMR spectroscopy and an S64V-EmrE mutant. The substrate-bound protein exhibits distinct structures at acidic and basic pH, reflecting changes upon binding or release of a proton from residue E14, respectively. To obtain insight into the protein dynamics that mediate substrate transport, here we measure 15N rotating-frame spin-lattice relaxation (R1ρ) rates of F4-TPP+-bound S64V-EmrE in lipid bilayers under magic-angle spinning (MAS). Using perdeuterated and back-exchanged protein and 1H-detected 15N spin-lock experiments under 55 kHz MAS, we measured 15N R1ρ rates site-specifically. Many residues show spin-lock field-dependent 15N R1ρ relaxation rates. This relaxation dispersion indicates the presence of backbone motions at a rate of about 6000 s-1 at 280 K for the protein at both acidic and basic pH. This motional rate is 3 orders of magnitude faster than the alternating access rate but is within the range estimated for substrate binding. We propose that these microsecond motions may allow EmrE to sample different conformations to facilitate substrate binding and release from the transport pore.

Microsecond motions probed by near-rotary-resonance R1\u03c115N MAS NMR experiments: the model case of protein overall-rocking in crystals

Solid-state near-rotary-resonance measurements of the spin–lattice relaxation rate in the rotating frame (R1ρ) is a powerful NMR technique for studying molecular dynamics in the microsecond time scale. The small difference between the spin-lock (SL) and magic-angle-spinning (MAS) frequencies allows sampling very slow motions, at the same time it brings up some methodological challenges. In this work, several issues affecting correct measurements and analysis of 15N R1ρ data are considered in detail. Among them are signal amplitude as a function of the difference between SL and MAS frequencies, “dead time” in the initial part of the relaxation decay caused by transient spin-dynamic oscillations, measurements under HORROR condition and proper treatment of the multi-exponential relaxation decays. The multiple 15N R1ρ measurements at different SL fields and temperatures have been conducted in 1D mode (i.e. without site-specific resolution) for a set of four different microcrystalline protein samples (GB1, SH3, MPD-ubiquitin and cubic-PEG-ubiquitin) to study the overall protein rocking in a crystal. While the amplitude of this motion varies very significantly, its correlation time for all four sample is practically the same, 30–50 μs. The amplitude of the rocking motion correlates with the packing density of a protein crystal. It has been suggested that the rocking motion is not diffusive but likely a jump-like dynamic process.

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.

Protein conformational dynamics studied by 15N and 1H R1ρ relaxation dispersion: Application to wild-type and G53A ubiquitin crystals

Solid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45–60kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a β-turn region in the wild-type protein. We use 15N R1ρ relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-1H R1ρ relaxation to obtain insight into dynamics. We show that while quantitative interpretation of 1H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.

NMR Relaxation Dispersion

NMR relaxation dispersion experiments are used to characterize millisecond and microsecond motions and the role of these motions in regulation of protein tyrosine phosphatases (PTPs) In a pair of mechanistically indistinguishable PTPs with catalytic rates that vary by 50‐fold these experiments indicate that catalytic activity is regulated by the rate of active site loop closure. Furthermore, it is observed that small molecule binding distant from the active site loop alters the kinetic rates of substrate catalysis as well as the dynamics of loop closure.

Microsecond Motion Modulates Ubiquitin Binding through an Allosteric Backbone/Side Chain Network

Motion is involved in a large number of protein functions. Relaxation dispersion (RD) NMR experiments sensitively probe microsecond to millisecond motions. We conducted an in-depth RD analysis of the backbone and side chain methyl groups of ubquitin. This survey showed a large number of atoms (>30) with microsecond fluctuations. These atoms are distributed throughout the structure. Strikingly, nearly all show the same exchange rate, which suggests that ubiquitin undergoes collective motion involving both the backbone and side chains. Furthermore, comparison of different methyl nuclei indicates that the nature of the side chain fluctuations is almost entirely due to changes in rotamer populations. Thus, collective microsecond backbone motion is coupled to redistribution of side chain rotamer populations through a mechanism we term “population shuffling”. We present a single collective mode of motion that yields a reaction coordinate corresponding to the relaxation dispersion data. The resulting model indicates that a localized conformational switch distant from the binding interface propagates changes throughout the structure. Analysis of crystal structures confirms this allosteric network and suggests that the microsecond motion modulates binding to particular interaction partners.

Population shuffling of protein conformations.

Motions play a vital role in the functions of many proteins. Discrete conformational transitions to excited states, happening on timescales of hundreds of microseconds, have been extensively characterized. On the other hand, the dynamics of the ground state are widely unexplored. Newly developed high-power relaxation dispersion experiments allow the detection of motions up to a one-digit microsecond timescale. These experiments showed that side chains in the hydrophobic core as well as at protein-protein interaction surfaces of both ubiquitin and the third immunoglobulin binding domain of protein G move on the microsecond timescale. Both proteins exhibit plasticity to this microsecond motion through redistribution of the populations of their side-chain rotamers, which interconvert on the picosecond to nanosecond timescale, making it likely that this "population shuffling" process is a general mechanism.

Population Shuffling of Protein Conformations

AbstractMotions play a vital role in the functions of many proteins. Discrete conformational transitions to excited states, happening on timescales of hundreds of microseconds, have been extensively characterized. On the other hand, the dynamics of the ground state are widely unexplored. Newly developed high‐power relaxation dispersion experiments allow the detection of motions up to a one‐digit microsecond timescale. These experiments showed that side chains in the hydrophobic core as well as at protein–protein interaction surfaces of both ubiquitin and the third immunoglobulin binding domain of protein G move on the microsecond timescale. Both proteins exhibit plasticity to this microsecond motion through redistribution of the populations of their side‐chain rotamers, which interconvert on the picosecond to nanosecond timescale, making it likely that this “population shuffling” process is a general mechanism.

Water scaffolding in collagen: Implications on protein dynamics as revealed by solid‐state NMR

Solid-state NMR studies of collagen samples of various origins confirm that the amplitude of collagen backbone and sidechain motions increases significantly on increasing the water content. This conclusion is supported by the changes observed in three different NMR observables: (i) the linewidth dependence on the 1H decoupling frequency; (ii) 13C CSA changes for the peptide carbonyl groups, and (iii) dephasing rates of 1H-13C dipolar couplings. In particular, a nearly threefold increase in motional amplitudes of the backbone librations about C-Cα or N-Cα bonds was found on increasing the added water content up to 47 wt%D2 O. On the basis of the frequencies of NMR observables involved, the timescale of the protein motions dependent on the added water content is estimated to be of the order of microseconds. This estimate agrees with that from wideline T2(1)H NMR measurements. Also, our wideline 1H NMR measurements revealed that the timescale of the microsecond motions in proteins reduces significantly on increasing the added water content, i.e., an ∼15-fold increase in protein motional frequencies is observed on increasing the added water content to 45 wt% D2 O. The observed changes in collagen dynamics is attributed to the increase in water translational diffusion on increasing the amount of added water, which leads to more frequent "bound water/free water" exchange on the protein surface, accompanied by the breakage and formation of new hydrogen bonds with polar functionalities of protein.

Interactions between PTB RRMs Induce Slow Motions and Increase RNA Binding Affinity

Polypyrimidine tract binding protein (PTB) participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site-mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA recognition motifs (RRM3 and RRM4), which interact with each other to form a stable complex (PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded structures that NMR data show to be similar to their structure in PRB1:34. The RNA binding properties of the domains differ dramatically. The affinity of each separate RRM for polypyrimidine tracts is far weaker than that of PTB1:34, and simply mixing the two RRMs does not create an equivalent binding platform. 15N NMR relaxation experiments show that PTB1:34 has slow, microsecond motions throughout both RRMs including the interdomain linker. This is in contrast to the individual domains, RRM3 and RRM4, where only a few backbone amides are flexible on this time scale. The slow backbone dynamics of PTB1:34, induced by packing of RRM3 and RRM4, could be essential for high-affinity binding to a flexible polypyrimidine tract RNA and also provide entropic compensation for its own formation.

Selective Characterization of Microsecond Motions in Proteins by NMR Relaxation

The three-dimensional structures of macromolecules fluctuate over a wide range of time-scales. Separating the individual dynamic processes according to frequency is of importance in relating protein motions to biological function and stability. We present here a general NMR method for the specific characterization of microsecond motions at backbone positions in proteins even in the presence of other dynamics such as large-amplitude nanosecond motions and millisecond chemical exchange processes. The method is based on measurement of relaxation rates of four bilinear coherences and relies on the ability of strong continuous radio frequency fields to quench millisecond chemical exchange. The utility of the methodology is demonstrated and validated through two specific examples focusing on the thermo-stable proteins, ubiquitin and protein L, where it is found that small-amplitude microsecond dynamics are more pervasive than previously thought. Specifically, these motions are localized to alpha helices, loop regions, and regions along the rim of beta sheets in both of the proteins examined. A third example focuses on a 28 kDa ternary complex of the chaperone Chz1 and the histones H2A.Z/H2B, where it is established that pervasive microsecond motions are localized to a region of the chaperone that is important for stabilizing the complex. It is further shown that these motions can be well separated from extensive millisecond dynamics that are also present and that derive from exchange of Chz1 between bound and free states. The methodology is straightforward to implement, and data recorded at only a single static magnetic field are required.

C-terminal domain of insulin-like growth factor (IGF) binding protein 6: conformational exchange and its correlation with IGF-II binding.

Insulin-like growth factor binding proteins (IGFBPs) function as carriers and regulators of the insulin-like growth factors (IGF-I and -II). Within the family of six binding proteins, IGFBP-6 is unique in having a 20-100-fold higher affinity for IGF-II over IGF-I and appears to act primarily as an inhibitor of IGF-II actions. We have recently determined the solution structure of the C-terminal domain of IGFBP-6 (C-BP-6), which shows the presence of substantial flexible regions, including three loop regions. In this paper, we report results from (15)N relaxation measurements carried out in both the laboratory and rotating frames. Analysis of conventional (15)N relaxation data (R(1), R(2), and steady-state (15)N-[(1)H] nuclear Overhauser effect) indicated that there was a considerable number of residues involved in conformational/chemical exchange. Measurements of off-resonance (15)N R(1)(rho) in the rotating frame and (15)N relaxation dispersion using an in- and antiphase coherence-averaged Carr-Purcell-Meiboom-Gill sequence were thus carried out to gain further insight into the solution dynamics of C-BP-6. Although the off-resonance (15)N relaxation data showed no clear evidence for residues undergoing microsecond motion, the (15)N relaxation dispersion data allowed us to identify 15 residues that clearly exhibit submilli- to millisecond motion. A good correlation was observed between residues exhibiting motion at submilli- to millisecond time scales and those affected by IGF-II binding, as identified through the perturbation of nuclear magnetic resonance (NMR) spectra of C-BP-6 following IGF-II addition. A complete NMR relaxation study of C-BP-6 dynamics in complex with IGF-II was hampered by peak broadening and disappearance of C-BP-6 in the presence of IGF-II. Nonetheless, current results strongly suggest possible conformation switching or population shifting between pre-existing conformations in C-BP-6 upon binding to IGF-II.

Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix.

We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.

Microsecond dynamics of biological macromolecules

Publisher Summary This chapter discusses that fluorescence spectroscopy is widely used to study the nanosecond timescale dynamics of biological macromolecules. The spectral observables are sensitive to nanosecond dynamics because emission also occurs on the nanosecond time scale. While nanosecond biopolymers dynamics are important, these rapid processes reflect mostly local fluorophore motions and its interactions with the immediate environment. However, biological macromolecules also display structural changes on the microsecond time scale. Processes that occur on the microsecond time scale include domain flexing in proteins and lateral diffusion in membranes. It is also likely that nucleic acid junctions and structured RNAs display microsecond motions. It discusses that fluorescence is now capable of detecting microsecond dynamics. This change in time scale is made possible by the development of metal-ligand complexes (MLCs), which display decay times ranging from 10 nsec to more than 10/μsec. The MLCs display several spectral characteristics that make them useful probes, including high photostability, a large Stokes shift, and polarized emission. The chapter presents data and simulations showing the possibility of measuring protein domain flexing, lateral diffusion in membranes, and microsecond rotational correlation times. Fluorescence is no longer trapped on the nanosecond time scale, and can be used to quantify dynamic processes from nanoseconds to microseconds to milliseconds because the lanthanides display millisecond decay times.

Microsecond motions of the lipids associated with trypsinized Na,K-ATPase membranes. Progressive saturation spin-label electron spin resonance studies.

The microsecond motions of spin-labeled lipids associated with the Na(+)/K(+)-transporting ATP hydrolase (Na,K-ATPase) in native and tryptically shaved membranes from Squalus acanthias have been studied by progressive saturation electron spin resonance (ESR). This includes both the segmental mobility of the lipid chains and the exchange dynamics of the lipids interacting directly with the protein. The lipids at the protein interface display a temperature-dependent chain mobility on the submicrosecond time scale. Exchange of these lipids with those in the bulk bilayer regions of the membrane takes place on the time scale of the nitroxide spin-lattice relaxation, i.e., in the microsecond regime. The off-rates for exchange directly reflect the specificity of ionized fatty acids relative to protonated fatty acids for interaction with the Na,K-ATPase. These essential features of the lipid dynamics at the intramembranous protein surface, namely, a temperature-dependent exchange on the microsecond time scale that reflects the lipid selectivity, are preserved on removing the extramembranous parts of the Na,K-ATPase by extensive trypsinization.

The mobility of troponin C and troponin I in muscle.

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca2+ concentration. The binding of Ca2+ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (tau r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg2+ by Ca2+ at these sites (tau r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca2+ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl.

Myosin head orientation and mobility during isometric contraction: effects of osmotic compression

We have correlated the mobility and the generation of force of myosin heads by applying radial compression to isometrically contracting muscle fibers. Osmotic pressure was produced by dextran T-500, and its effect on the orientation and mobility of myosin heads labeled with N-(1-oxy-2,2,5,5-tetramethyl-4-pyperidinyl)maleimide was observed by conventional and saturation-transfer electron paramagnetic resonance methods. A biphasic behavior is spectral changes coinciding with the tension dependence was observed as the fibers were compressed. At diameters above the equilibrium spacing, the large myosin head disorder characteristic during contraction in the absence of compression was largely maintained, whereas the mobility decreased threefold, from tauR approximately 25 microseconds to approximately 80-90 microseconds. The inhibition of fast microsecond motions was not accompanied by tension loss, implying that these motions are not necessary for force generation. At diameters below the equilibrium spacing, both the disorder and the mobility decreased dramatically in parallel with the tension inhibition, suggesting that slower microsecond motions and the disorder of the myosin head are necessary for muscle function.

Myosin catalytic domain flexibility in MgADP.

Conventional EPR studies of muscle fibers labeled with a novel alpha-iodoketo spin label at Cys-707 of the myosin head revealed substantial internal domain reorganization on the addition of ADP to rigor fibers. The spin probes that are well-ordered in the rigor state become disordered and form two distinct populations. These orientational changes do not correspond to rotation of the myosin catalytic domain as a whole because other probes (maleimide and iodoacetamide nitroxides attached to the same Cys-707 of myosin head) report only a small (5-10 degrees) torsional rotation and little or no change in the tilt angle [Ajtai et al. (1992) Biochemistry 31, 207-17; Fajer (1994) Biophys. J. 66, 2039-50]. In the presence of ADP, the labeled domain becomes more flexible and executes large-amplitude microsecond motions, as measured by saturation-transfer EPR with rates (tau r = 150 microseconds) intermediate between the rotations of detached (tau r = 7 microseconds) and rigor heads (tau r = 2500 microseconds). This finding contrasts with an absence of global motion of the myosin head in ADP (tau r = 2200 microseconds) as reported by the maleimide spin label. Our results imply that the myosin head in a single chemical state (AM.ADP) is capable of attaining many internal configurations, some of which are dynamic. The presence of these slow structural fluctuations might be related to the slow release of the hydrolysis products of actomyosin ATPase.

Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers

We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)