Microsecond-millisecond Time Scale Research Articles

Perspective: How Fast Dynamics Affect Slow Function in Protein Machines.

Internal motions in proteins take place on a broad range of time- and space-scales. The potential roles of these dynamics in the biochemical functions of proteins have intrigued biophysicists for many years, and multiple mechanisms to couple motions to function have been proposed. Some of these mechanisms have relied on equilibrium concepts. For example, the modulation of dynamics was proposed to change the entropy of a protein, hence affecting processes such as binding. This so-called dynamic allostery scenario has been demonstrated in several recent experiments. Perhaps even more intriguing may be models that involve out-of-equilibrium operation, which by necessity require the input of energy. We discuss several recent experimental studies that expose such potential mechanisms for coupling dynamics and function. In Brownian ratchets, for example, directional motion is promoted by switching a protein between two free energy surfaces. An additional example involves the effect of microsecond domain-closure dynamics of an enzyme on its much slower chemical cycle. These observations lead us to propose a novel two-time-scale paradigm for the activity of protein machines: fast equilibrium fluctuations take place on the microsecond-millisecond time scale, while on a slower time scale, free energy is invested in order to push the system out of equilibrium and drive functional transitions. Motions on the two time scales affect each other and are essential for the overall function of these machines.

Solution structure ensemble of human obesity-associated protein FTO reveals druggable surface pockets at the interface between the N- and C-terminal domain

The fat mass and obesity-associated FTO protein catalyzes demethylation of the N6-methyladenosine, an epigenetic mark that controls several metabolic pathways by modulating the transcription, translation, and cellular localization of RNA molecules. Since the discovery that its overexpression links to the development of obesity and cancer, FTO was the target of screening campaigns and structure-based drug design efforts. Although several FTO inhibitors were generated, these often lack potency or selectivity. Herein, we investigate the structure and dynamics of human FTO in solution. We show that the structure of the catalytic N-terminal domain is unstable in the absence of the C-terminal domain, which explains why the isolated N-terminal domain is incompetent for catalysis and suggests that the domain interaction represents a target for the development of specific inhibitors. Then, by using NMR relaxation measurements, we show that the interface between the FTO structural domains, the active site, and several peripheral loops undergo conformational dynamics on both the picosecond–nanosecond and microsecond–millisecond timescales. Consistent with this, we found that the backbone amide residual dipolar couplings measured for FTO in phage pf1 are inconsistent with the static crystal structure of the enzyme. Finally, we generated a conformational ensemble for apo FTO that satisfies the solution NMR data by combining the experimental residual dipolar couplings with accelerated molecular dynamics simulations. Altogether, the structural ensemble reported in this work provides an atomic-resolution model of apo FTO and reveals transient surface pockets at the domain interface that represent potential targets for the design of allosteric inhibitors.

Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and \u0394N6 \u03b22-Microglobulin

Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochemical oxidation of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β2-microglobulin (β2m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimised LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures.Graphical ᅟ

Internal Electric Field Modulation in Molecular Electronic Devices by Atmosphere and Mobile Ions.

The internal potential profile and electric field are major factors controlling the electronic behavior of molecular electronic junctions consisting of ∼1-10 nm thick layers of molecules oriented in parallel between conducting contacts. The potential profile is assumed linear in the simplest cases, but can be affected by internal dipoles, charge polarization, and electronic coupling between the contacts and the molecular layer. Electrochemical processes in solutions or the solid state are entirely dependent on modification of the electric field by electrolyte ions, which screen the electrodes and form the ionic double layers that are fundamental to electrode kinetics and widespread applications. The current report investigates the effects of mobile ions on nominally solid-state molecular junctions containing aromatic molecules covalently bonded between flat, conducting carbon surfaces, focusing on changes in device conductance when ions are introduced into an otherwise conventional junction design. Small changes in conductance were observed when a polar molecule, acetonitrile, was present in the junction, and a large decrease of conductance was observed when both acetonitrile (ACN) and lithium ions (Li+) were present. Transient experiments revealed that conductance changes occur on a microsecond-millisecond time scale, and are accompanied by significant alteration of device impedance and temperature dependence. A single molecular junction containing lithium benzoate could be reversibly transformed from symmetric current-voltage behavior to a rectifier by repetitive bias scans. The results are consistent with field-induced reorientation of acetonitrile molecules and Li+ ion motion, which screen the electrodes and modify the internal potential profile and provide a potentially useful means to dynamically alter junction electronic behavior.

Conformational Flexibility Differentiates Naturally Occurring Bet v 1 Isoforms.

The protein Bet v 1 represents the main cause for allergic reactions to birch pollen in Europe and North America. Structurally homologous isoforms of Bet v 1 can have different properties regarding allergic sensitization and Th2 polarization, most likely due to differential susceptibility to proteolytic cleavage. Using NMR relaxation experiments and molecular dynamics simulations, we demonstrate that the initial proteolytic cleavage sites in two naturally occurring Bet v 1 isoforms, Bet v 1.0101 (Bet v 1a) and Bet v 1.0102 (Bet v 1d), are conformationally flexible. Inaccessible cleavage sites in helices and strands are highly flexible on the microsecond-millisecond time scale, whereas those located in loops display faster nanosecond-microsecond flexibility. The data consistently show that Bet v 1.0102 is more flexible and conformationally heterogeneous than Bet v 1.0101. Moreover, NMR hydrogen-deuterium exchange measurements reveal that the backbone amides in Bet v 1.0102 are significantly more solvent exposed, in agreement with this isoform’s higher susceptibility to proteolytic cleavage. The differential conformational flexibility of Bet v 1 isoforms, along with the transient exposure of inaccessible sites to the protein surface, may be linked to proteolytic susceptibility, representing a potential structure-based rationale for the observed differences in Th2 polarization and allergic sensitization.

STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism.

START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.

A 15N CPMG relaxation dispersion experiment more resistant to resonance offset and pulse imperfection

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion is a powerful NMR method to study protein dynamics on the microsecond–millisecond time scale. J-coupling, resonance offset, radio frequency field inhomogeneity, and pulse imperfection often introduce systematic errors into the measured transverse relaxation rates. Here we proposed a modified continuous wave decoupling CPMG experiment, which is more unaffected by resonance offset and pulse imperfection. We found that it is unnecessary to match the decoupling field strength with the delay between CPMG refocusing pulses, provided that decoupling field is strong enough. The performance of the scheme proposed here was shown by simulations and further demonstrated experimentally on a fatty acid binding protein.

Enzyme dynamics from NMR spectroscopy.

ConspectusBiological activities of enzymes, including regulation or coordination of mechanistic stages preceding or following the chemical step, may depend upon kinetic or equilibrium changes in protein conformations. Exchange of more open or flexible conformational states with more closed or constrained states can influence inhibition, allosteric regulation, substrate recognition, formation of the Michaelis complex, side reactions, and product release. NMR spectroscopy has long been applied to the study of conformational dynamic processes in enzymes because these phenomena can be characterized over multiple time scales with atomic site resolution. Laboratory-frame spin-relaxation measurements, sensitive to reorientational motions on picosecond–nanosecond time scales, and rotating-frame relaxation-dispersion measurements, sensitive to chemical exchange processes on microsecond–millisecond time scales, provide information on both conformational distributions and kinetics. This Account reviews NMR spin relaxation studies of the enzymes ribonuclease HI from mesophilic (Escherichia coli) and thermophilic (Thermus thermophilus) bacteria, E. coli AlkB, and Saccharomyces cerevisiae triosephosphate isomerase to illustrate the contributions of conformational flexibility and dynamics to diverse steps in enzyme mechanism.Spin relaxation measurements and molecular dynamics (MD) simulations of the bacterial ribonuclease H enzymes show that the handle region, one of three loop regions that interact with substrates, interconverts between two conformations. Comparison of these conformations with the structure of the complex between Homo sapiens ribonuclease H and a DNA:RNA substrate suggests that the more closed state is inhibitory to binding. The large population of the closed conformation in T. thermophilus ribonuclease H contributes to the increased Michaelis constant compared with the E. coli enzyme. NMR spin relaxation and fluorescence spectroscopy have characterized a conformational transition in AlkB between an open state, in which the side chains of methionine residues in the active site are disordered, and a closed state, in which these residues are ordered. The open state is highly populated in the AlkB/Zn(II) complex, and the closed state is highly populated in the AlkB/Zn(II)/2OG/substrate complex, in which 2OG is the 2-oxoglutarate cosubstrate and the substrate is a methylated DNA oligonucleotide. The equilibrium is shifted to approximately equal populations of the two conformations in the AlkB/Zn(II)/2OG complex. The conformational shift induced by 2OG ensures that 2OG binds to AlkB/Zn(II) prior to the substrate. In addition, the opening rate of the closed conformation limits premature release of substrate, preventing generation of toxic side products by reaction with water. Closure of active site loop 6 in triosephosphate isomerase is critical for forming the Michaelis complex, but reopening of the loop after the reaction is (partially) rate limiting. NMR spin relaxation and MD simulations of triosephosphate isomerase in complex with glycerol 3-phosphate demonstrate that closure of loop 6 is a highly correlated rigid-body motion. The MD simulations also indicate that motions of Gly173 in the most flexible region of loop 6 contribute to opening of the active site loop for product release. Considered together, these three enzyme systems illustrate the power of NMR spin relaxation investigations in providing global insights into the role of conformational dynamic processes in the mechanisms of enzymes from initial activation to final product release.

Chromophore Photoreduction in Red Fluorescent Proteins Is Responsible for Bleaching and Phototoxicity

Red fluorescent proteins (RFPs) are indispensable tools for deep-tissue imaging, fluorescence resonance energy transfer applications, and super-resolution microscopy. Using time-resolved optical spectroscopy this study investigated photoinduced dynamics of three RFPs, KillerRed, mRFP, and DsRed. In all three RFPs, a new transient absorption intermediate was observed, which decays on a microsecond–millisecond time scale. This intermediate is characterized by red-shifted absorption at 1.68–1.72 eV (λmax = 720–740 nm). On the basis of electronic structure calculations, experimental evidence, and published literature, the chemical nature of the intermediate is assigned to an unusual open-shell dianionic chromophore (dianion-radical) formed via photoreduction. A doubly charged state that is not stable in the isolated (gas phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.

Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection

Protein dynamics on the microsecond–millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the backbone amides or side-chain methyl groups, while experiments targeting other sites have been used more sparingly. Aromatic side chains are useful probes of protein dynamics, because they are over-represented in protein binding interfaces, have important catalytic roles in enzymes, and form a sizable part of the protein interior. Here we present an off-resonance R 1ρ experiment for measuring microsecond to millisecond conformational exchange of aromatic side chains in selectively 13C labeled proteins by means of longitudinal- and transverse-relaxation optimization. Using selective excitation and inversion of the narrow component of the 13C doublet, the experiment achieves significant sensitivity enhancement in terms of both signal intensity and the fractional contribution from exchange to transverse relaxation; additional signal enhancement is achieved by optimizing the longitudinal relaxation recovery of the covalently attached 1H spins. We validated the L-TROSY-selected R 1ρ experiment by measuring exchange parameters for Y23 in bovine pancreatic trypsin inhibitor at a temperature of 328 K, where the ring flip is in the fast exchange regime with a mean waiting time between flips of 320 μs. The determined chemical shift difference matches perfectly with that measured from the NMR spectrum at lower temperatures, where separate peaks are observed for the two sites. We further show that potentially complicating effects of strong scalar coupling between protons (Weininger et al. in J Phys Chem B 117: 9241–9247, 2013b) can be accounted for using a simple expression, and provide recommendations for data acquisition when the studied system exhibits this behavior. The present method extends the repertoire of relaxation methods tailored for aromatic side chains by enabling studies of faster processes and improved control over artifacts due to strong coupling.

NMR Investigation of Chloromethane Complexes of Cryptophane-A and Its Analogue with Butoxy Groups

Host–guest complexes between cryptophane-A as host and dichloromethane and chloroform as guests are investigated using 1H and 13C NMR spectroscopy. Moreover, a related cryptophane, with the methoxy groups replaced by butoxy units (cryptophane-But), and its complexes with the same guests were also studied. Variable temperature spectra showed effects of chemical exchange between the free and bound guests, as well as of conformational exchange of the host. The guest exchange was studied quantitatively by exchange spectroscopy or line shape analysis. Extraction of kinetic and thermodynamic parameters led to the characterization of the affinity between guests and hosts. On the other hand, the host exchange was investigated by means of 13C Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion which aims at the determination of the transverse relaxation rate R2, the inverse of the transverse relaxation time T2, as a function of the repetition of the π pulses in a CPMG train. The variation of the measured transverse relaxation rate with the repetition rate νCPMG indicated conformational exchange occurring on the microsecond–millisecond time scale. Structural information was obtained through measurements of cross-relaxation rates, both within the host and between the host and the guest protons. The NMR results were supported by DFT calculations.

Internal Motions Prime cIAP1 for Rapid Activation

Cellular inhibitor of apoptosis 1 (cIAP1) is an E3 ubiquitin ligase with roles in both apoptosis and NF-κB signaling. Recent crystallographic and SAXS data have shown that the ligase activity of cIAP1 is autoinhibited by occlusion of the RING dimerization interface through several inter-domain interactions, most critically in the BIR3:RING interface. This interaction is disrupted by SMAC and SMAC mimetics, initiating cIAP1 dimerization through its newly exposed RING domain and activation of its E3 ligase activity. Activation of cIAP1's ligase activity leads to cIAP1 autoubiquitination and proteasomal degradation. Intriguingly, we observed that although the binding site for cIAP1's E2 partner enzyme is occluded in the crystal structure, E2 binds to both the monomeric and dimeric forms of cIAP1 with nearly the same affinity. This unexpected result indicated that there were likely significant residual motions occurring within monomeric cIAP1. Through NMR relaxation dispersion measurements and resonance broadening we have been able to detect significant motions occurring on relatively fast, microsecond-millisecond timescales at specific domain interfaces present in the autoinhibited form of cIAP1. In addition, time-resolved SAXS measurements conducted in line with a stopped flow injection system have allowed us to measure the opening rate of cIAP1, which occurs over hundreds of milliseconds. The tight regulation of apoptosis is essential in multicellular organisms and the proteins involved in transducing pro-apoptotic and anti-apoptotic signals are no doubt under stringent evolutionary control. The internal motions present in cIAP1 likely arose from the competing pressures to activate apoptosis as quickly as possible while maintaining the fidelity of the pro-apoptotic signal.

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J–K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to target specific dynamic states of the Eph receptor LBDs.

Crystallographic and NMR Evidence for Flexibility in Oligosaccharyltransferases and Its Catalytic Significance

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the+2 position in theN-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains the+2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.

Dynamics and activation in response regulators: the β4-α4 loop

Two-component signal transduction systems of microbes are a primary means to respond to signals emanating from environmental and metabolic fluctuations as well as to signals coordinating the cell cycle with macromolecular syntheses, among a large variety of other essential roles. Signals are recognized by a sensor domain of a histidine kinase which serves to convert signal binding to an active transmissible phosphoryl group through a signal-induced ATP-dependent autophosphorylation reaction directed to histidine residue. The sensor kinase is specifically mated to a response regulator, to which it transfers the phosphoryl group that activates the response regulator's function, most commonly gene repression or activation but also interaction with other regulatory proteins. Two-component systems have been genetically amplified to control a wide variety of cellular processes; for example, both Escherichia coli and Pseudomonas aeruginosa have 60 plus confirmed and putative two-component systems. Bacillus subtilis has 30 plus and Nostoc punctiformis over 100. As genetic amplification does not result in changes in the basic structural folds of the catalytic domains of the sensor kinase or response regulators, each sensor kinase must recognize its partner through subtle changes in residues at the interaction surface between the two proteins. Additionally, the response regulator must prepare itself for efficient activation by the phosphorylation event. In this short review, we discuss the contributions of the critical β4-α4 recognition loop in response regulators to their function. In particular, we focus on this region's microsecond-millisecond timescale dynamics propensities and discuss how these motions play a major role in response regulator recognition and activation.

Conformational Conversion during Amyloid Formation at Atomic Resolution

SummaryNumerous studies of amyloid assembly have indicated that partially folded protein species are responsible for initiating aggregation. Despite their importance, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they cause aggregation remain elusive. Here, we use ΔN6, a truncation variant of the naturally amyloidogenic protein β2-microglobulin (β2m), to determine the solution structure of a nonnative amyloidogenic intermediate at high resolution. The structure of ΔN6 reveals a major repacking of the hydrophobic core to accommodate the nonnative peptidyl-prolyl trans-isomer at Pro32. These structural changes, together with a concomitant pH-dependent enhancement in backbone dynamics on a microsecond-millisecond timescale, give rise to a rare conformer with increased amyloidogenic potential. We further reveal that catalytic amounts of ΔN6 are competent to convert nonamyloidogenic human wild-type β2m (Hβ2m) into a rare amyloidogenic conformation and provide structural evidence for the mechanism by which this conformational conversion occurs.

Conformational flexibility of Y145Stop human prion protein amyloid fibrils probed by solid-state nuclear magnetic resonance spectroscopy.

Amyloid aggregates of a C-truncated Y145Stop mutant of human prion protein, huPrP23-144, associated with a heritable amyloid angiopathy, have previously been shown to contain a compact, relatively rigid, and beta-sheet-rich approximately 30-residue amyloid core near the C-terminus under physiologically relevant conditions. In contrast, the remaining huPrP23-144 residues display considerable conformational dynamics, as evidenced by the absence of corresponding signals in cross-polarization (CP)-based solid-state NMR (SSNMR) spectra under ambient conditions and their emergence in analogous spectra recorded at low temperature on frozen fibril samples. Here, we present the direct observation of residues comprising the flexible N-terminal domain of huPrP23-144 amyloid by using 2D J-coupling-based magic-angle spinning (MAS) SSNMR techniques. Chemical shifts for these residues indicate that the N-terminal domain is effectively an ensemble of protein chains with random-coil-like conformations. Interestingly, a detailed analysis of signal intensities in CP-based 3D SSNMR spectra suggests that non-negligible molecular motions may also be occurring on the NMR time scale within the relatively rigid core of huPrP23-144 amyloid. To further investigate this hypothesis, quantitative measurements of backbone dipolar order parameters and transverse spin relaxation rates were performed for the core residues. The observed order parameters indicate that, on the submicrosecond time scale, these residues are effectively rigid and experience only highly restricted and relatively uniform motions similar to those characteristic for well-structured regions of microcrystalline proteins. On the other hand, significant variations in magnitude of transverse spin relaxation rates were noted for residues present at different locations within the core region and correlated with observed differences in spectral intensities. While interpreted only qualitatively at the present time, the extent of the observed variations in transverse relaxation rates is consistent with the presence of relatively slow, microsecond-millisecond time scale chemical exchange type phenomena within the huPrP23-144 amyloid core.

NMR Investigation of Tyr105 Mutants in TEM-1 β-Lactamase: DYNAMICS ARE CORRELATED WITH FUNCTION

The existence of coupled residue motions on various time scales in enzymes is now well accepted, and their detailed characterization has become an essential element in understanding the role of dynamics in catalysis. To this day, a handful of enzyme systems has been shown to rely on essential residue motions for catalysis, but the generality of such phenomena remains to be elucidated. Using NMR spectroscopy, we investigated the electronic and dynamic effects of several mutations at position 105 in TEM-1 beta-lactamase, an enzyme responsible for antibiotic resistance. Even in absence of substrate, our results show that the number and magnitude of short and long range effects on (1)H-(15)N chemical shifts are correlated with the catalytic efficiencies of the various Y105X mutants investigated. In addition, (15)N relaxation experiments on mutant Y105D show that several active-site residues of TEM-1 display significantly altered motions on both picosecond-nanosecond and microsecond-millisecond time scales despite many being far away from the site of mutation. The altered motions among various active-site residues in mutant Y105D may account for the observed decrease in catalytic efficiency, therefore suggesting that short and long range residue motions could play an important catalytic role in TEM-1 beta-lactamase. These results support previous observations suggesting that internal motions play a role in promoting protein function.

An investigation of the dynamics of spermine bound to duplex and quadruplex DNA by 13C NMR spectroscopy

A detailed analysis of the (13)C relaxation of (13)C-labelled spermine bound to duplex and quadruplex DNA is presented. T(1), T(2) and heteronuclear NOE data were collected at four (13)C frequencies (75.4, 125.7, 150.9 and 201.2 MHz). The data were analyzed in terms of a frequency-dependent order parameter, S (2)(omega), to estimate the generalized order parameter and the contributions to the relaxation from different motional frequencies in the picosecond-nanosecond timescale and from any exchange processes that may be occurring on the microsecond-millisecond timescale. The relaxation data was surprisingly similar for spermine bound to two different duplexes and a linear parallel quadruplex. Analysis of the relaxation data from these complexes confirmed the conclusions of previous studies that the dominant motion of spermine is independent of the macroscopic tumbling of the DNA and has an effective correlation time of approximately 50 ps. In contrast, spermine bound to a folded antiparallel quadruplex had faster relaxation rates, especially R (2). As with the other complexes, a fast internal motion of the order of 50 ps makes a substantial contribution to the relaxation. The generalized order parameter for spermine bound to duplex DNA and the linear quadruplex is small but is larger for spermine bound to the folded quadruplex. In the latter case, there is evidence for exchange between at least two populations of spermine occurring on the microsecond-millisecond timescale.

Structural Insight into the Binding Diversity between the Tyr-phosphorylated Human EphrinBs and Nck2 SH2 Domain

The binding interaction between the Nck2 SH2 domain and the phosphorylated ephrinB initiates a critical pathway for the reverse signaling network mediated by Eph receptor-ephrinB. Previously, the NMR structure and Tyr phosphorylations of the human ephrinB cytoplasmic domain have been studied. To obtain a complete story, it would be of significant interest to determine the structure of the Nck2 SH2 domain that shows a low sequence identity to other SH2 domains with known structures. Here, we report the determination of the solution structure of the human Nck2 SH2 domain and investigate its interactions with three phosphorylated ephrinB fragments by NMR spectroscopy. The results indicate that: 1) although the human Nck2 SH2 domain adopts a core tertiary fold common to all SH2 domains, it owns some unique properties such as a shorter C-terminal helix and unusual electrostatic potential surface. However, the most striking finding is that the C-terminal tail of the human Nck2 SH2 domain adopts a short antiparallel beta-sheet that, to the best of our knowledge, has never been identified in other SH2 domains. The truncation study suggests that one function of the C-terminal tail is to control the folding/solubility of the SH2 domain. 2) In addition to [Tyr(P)304]ephrinB2(301-322) and [Tyr(P)316]ephrinB2(301-322), here we identified [Tyr(P)330]ephrinB2(324-333) also capable of binding to the SH2 domain. The detailed NMR study indicated that the binding mechanisms for the three ephrinB fragments might be different. The binding with [Tyr(P)304]-ephrinB2(301-322) and [Tyr(P)316]ephrinB2(301-322) might be mostly involved in the residues over the N-half of the SH2 domain and provoked a significant increase in the backbone and side chain dynamics of the SH2 domain on the microsecond-millisecond time scale. In contrast, binding with [Tyr(P)330]ephrinB2(324-333) might have most residues over both halves engaged but induced less profound conformational dynamics on the mus-ms time scale.