Conformational Exchange Processes Research Articles

Unexpected dynamics in femtomolar complexes of binding proteins with peptides

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

Cis-trans isomerization in cyclosporin C dissolved in acetonitrile

Cyclosporin is an 11-amino acid cyclic peptide with pharmacologically valuable properties which has a variety of actual and potential applications. Its activity relies on the cell membrane permeability which, in turn, depends on the structure of cyclosporin and its ability to change the conformation. In this work, conformational exchange processes occurring in cyclosporin C were studied using one- and two-dimensional nuclear magnetic resonance spectroscopy. The free energy barrier separating two major conformers observed in polar solution (acetonitrile) was found to be 77 ± 2 kJ/mol. Less populated conformation states are also present in the solution, which agrees with the ease of formation of multiple forms revealed by MD simulations of cyclosporin C.

Microsecond MD Simulations of the Plexin-B1 RBD: 2. N-H Probability Densities and Conformational Entropy in Ligand-Free, Rac1-Bound, and Dimer RBD.

Orientational probability densities, Peq = exp(-u) (u, local potential), of bond-vectors in proteins provide information on structural flexibility. The related conformational entropy, Sk = -∫Peq(ln Peq)dΩ - ln ∫dΩ, provides the entropic contribution to the free energy of the physical/biological process studied. We have developed a new method for deriving Peq and Sk from MD simulations, using the N-H bond as probe. Recently we used it to study the dimerization of the Rho GTPase binding domain of Plexin-B1 (RBD). Here we use it to study RBD binding to the small GTPase Rac1. In both cases 1 μs MD simulations have been employed. The RBD has the ubiquitin fold with four mostly long loops. L3 is associated with GTPase binding, L4 with RBD dimerization, L2 participates in interdomain interactions, and L1 has not been associated with function. We find that RBD-Rac1 binding renders L1, L3, and L4 more rigid and the turns β2/α1 and α2/β5 more flexible. By comparison, RBD dimerization renders L4 more rigid, and the α-helices, the β-strands, and L2 more flexible. The rigidity of L1 in RBDRAC is consistent with L1-L3 contacts seen in previous MD simulations. The analysis of the L3-loop reveals two states of distinct flexibility which we associate with involvement in slow conformational exchange processes differing in their rates. Overall, the N-H bonds make an unfavorable entropic contribution of (5.9 ± 0.9) kJ/mol to the free energy of RBD-Rac1 binding; they were found to make a favorably contribution of (-7.0 ± 0.7) kJ/mol to the free energy of RBD dimerization. In summary, the present study provides a new perspective on the impact of Rac1 binding and dimerization on the flexibility characteristics of the RBD. Further studies are stimulated by the results of this work.

Observation of Arginine Side-Chain Motions Coupled to the Global Conformational Exchange Process in Deubiquitinase A

Coupled motions have been demonstrated to be functionally important in a number of enzymes. Noncovalent side-chain interactions play essential roles in coordinating the motions across different structural elements in a protein. However, most of the dynamic studies of proteins are focused on backbone amides or methyl groups in the side chains and little is known about the polar and charged side chains. We have previously characterized the conformational dynamics of deubiquitinase A (DUBA), an isopeptidase, on the microsecond-to-millisecond (μs–ms) time scales with the amide 1H Carr–Purcell–Meiboom–Gill (CPMG) experiment. We detected a global conformational exchange process on a time scale of approximately 200 μs, which involves most of the structural elements in DUBA, including the active site and the substrate binding interface. Here, we extend our previous study on backbone amides to the arginine side-chain Nε–Hε groups using a modified 1H CPMG pulse sequence that can efficiently detect both backbone amide and arginine side-chain Nε–Hε signals in a single experiment. We found that the side chains of three arginines display motions on the same time scale as the backbone amides. Mutations of two of the three arginines to alanines result in a decrease in enzyme activity. One of these two arginines is located in a loop involved in substrate binding. This loop is not visible in the backbone amide-detected experiments due to excess line broadening induced by motions on the μs–ms time scales. These results clearly demonstrate that the motions of some arginine side chains are coupled to the global conformational exchange process and provide an additional probe for motions in a functionally important loop that did not yield visible backbone amide signals, suggesting the value of side-chain experiments on DUBA. The modified 1H CPMG pulse sequence allows the simultaneous characterization of backbone and arginine side-chain dynamics without any increase in data acquisition time and can be applied to the dynamic studies of any protein that displays measurable amide 1H relaxation dispersion.

Analysis of conformational exchange processes using methyl-TROSY-based Hahn echo measurements of quadruple-quantum relaxation.

Transverse nuclear spin relaxation is a sensitive probe of chemical exchange on timescales on the order of microseconds to milliseconds. Here we present an experiment for the simultaneous measurement of the relaxation rates of two quadruple-quantum transitions in CH-labelled methyl groups. These coherences are protected against relaxation by intra-methyl dipolar interactions and so have unexpectedly long lifetimes within perdeuterated biomacromolecules. However, these coherences also have an order of magnitude higher sensitivity to chemical exchange broadening than lower order coherences and therefore provide ideal probes of dynamic processes. We show that analysis of the static magnetic field dependence of zero-, double- and quadruple-quantum Hahn echo relaxation rates provides a robust indication of chemical exchange and can determine the signed relative magnitudes of proton and carbon chemical shift differences between ground and excited states. We also demonstrate that this analysis can be combined with established Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion measurements, providing improved precision in parameter estimates, particularly in the determination of H chemical shift differences.

Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis.

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole-3-glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde-3-phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate- and products-bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate-bound to the products-bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit.

Monitoring Conformational Changes in an Enzyme Conversion Inhibitor Using Pure Shift Exchange NMR Spectroscopy.

We report the acquisition of 2D NMR EXSY spectra with ultrahigh resolution, which allows for probing the slow conformational exchange process in a pharmaceutical compound. The resolution enhancement is achieved by implementing interferogram based PSYCHE homonuclear decoupling to generate a pure shift proton spectrum along the direct domain of the resulting data. The performance of this pure shift EXSY pulse sequence is compared to the standard experiment recorded under identical conditions. It is found that although being less sensitive and requiring a longer acquisition time, the quality of pure shift spectra allows for extracting exchange rates values that are coherent with the ones determined by standard approach, on a temperature range that demonstrates the robustness of the chosen homonuclear decoupling method. The resolution enhancement provided by the simplification of proton line shape allows for probing a higher number of proton sites whose analysis would have been biased using a standard method. These results open the way to a thorough and accurate study of chemical exchange processes based on a multi-site analysis of 2D pure shift EXSY spectra.

Frequency selective coherence transfer NMR spectroscopy to study the structural dynamics of high molecular weight proteins.

Multidimensional nuclear magnetic resonance (NMR) spectroscopy has enabled detailed characterizations of protein structures and dynamics that are closely linked to functions. However, it leads to a large sensitivity loss in applications to high molecular weight proteins, which is caused by spin relaxation during the frequency discrimination period in the indirect dimension. Here, we describe a selective coherence transfer scheme, which enables us to selectively observe 1H nuclei bonded to 15N or 13C nuclei with specified resonance frequencies. By utilizing this scheme, we achieved a 2.5- to 6-fold increase in signal height per unit of time with this scheme by avoiding the relaxation loss in the indirect dimension, as compared to the conventional two-dimensional heteronuclear correlation spectroscopy. We also demonstrated the effectiveness of this approach with applications to the membrane protein KirBac1.1, and characterized the functionally relevant conformational exchange process in both detergent micelles and a reconstituted membrane environment, corresponding to the apparent molecular masses of 220 kDa and 300 kDa, respectively.

Different modes of barrel opening suggest a complex pathway of ligand binding in human gastrotropin.

Gastrotropin, the intracellular carrier of bile salts in the small intestine, binds two ligand molecules simultaneously in its internal cavity. The molecular rearrangements required for ligand entry are not yet fully clear. To improve our understanding of the binding process we combined molecular dynamics simulations with previously published structural and dynamic NMR parameters. The resulting ensembles reveal two distinct modes of barrel opening with one corresponding to the transition between the apo and holo states, whereas the other affecting different protein regions in both ligation states. Comparison of the calculated structures with NMR-derived parameters reporting on slow conformational exchange processes suggests that the protein undergoes partial unfolding along a path related to the second mode of the identified barrel opening motion.

Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops β5-α3 and α3-β6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated “dark” conformational state.

Exchangeable deuterons introduce artifacts in amide 15N CEST experiments used to study protein conformational exchange.

Protein molecules sample different conformations in solution and characterizing these conformations is crucial to understanding protein function. 15N CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a 'visible' major state and 'invisible' minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope effect on the amide 15N chemical shifts. However at moderately high temperatures (~ 50°C) that are sometimes required to populate protein minor conformers to levels (~ 1%) that can be detected by CEST experiments solvent H/D exchange can lead to 'dips' in low B115N CEST profiles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of ~ 18kDa T4 Lysozyme (T4L) at 50°C and the ~ 11kDa E. coli hibernation promoting factor (HPF) at 52°C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D2O lock or by using a small amount (~ 1-3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer.

Microsecond Protein Dynamics from Combined Bloch-McConnell and Near-Rotary-Resonance R1p Relaxation-Dispersion MAS NMR.

Studying protein dynamics on microsecond-to-millisecond (μs-ms) time scales can provide important insight into protein function. In magic-angle-spinning (MAS) NMR, μs dynamics can be visualized by rotating-frame relaxation dispersion experiments in different regimes of radio-frequency field strengths: at low RF field strength, isotropic-chemical-shift fluctuation leads to "Bloch-McConnell-type" relaxation dispersion, while when the RF field approaches rotary resonance conditions bond angle fluctuations manifest as increased rate constants ("Near-Rotary-Resonance Relaxation Dispersion", NERRD). Here we explore the joint analysis of both regimes to gain comprehensive insight into motion in terms of geometric amplitudes, chemical-shift changes, populations and exchange kinetics. We use a numerical simulation procedure to illustrate these effects and the potential of extracting exchange parameters, and apply the methodology to the study of a previously described conformational exchange process in microcrystalline ubiquitin.

Spatial Structure and Conformational Mobility of Seven-Membered Cyclic Acetals and Ketals Containing Pyridoxine Moiety in Solution by NMR Methods

In the last decade, pyridoxine derivatives were widely used for the synthesis of molecules with important biological and physical properties. However, rational synthesis of the compounds with desirable biochemical or physical properties often requires information about their spatial structure and conformational mobility. NMR spectroscopy is a powerful tool for conformational analysis of biologically important samples, such as pyridoxine derivatives, in solution. This paper is devoted to a review of the results obtained by our group over the last 5 years on the NMR study of nine newly synthesized pyridoxine derivatives in acetone solution. It was shown that studied compounds are involved into several conformational exchange processes. The activation energies of all observed conformational exchange processes were calculated.

Interplay between the Conformational Flexibility and Photoluminescent Properties of Mononuclear Pyridinophanecopper(I) Complexes.

The macrocyclic ligand conformational behavior in solution, solid-state structures and the photophysical properties of copper(I) cationic and neutral mononuclear complexes supported by tetradentate N, N'-dialkyl-2,11-diaza[3.3](2,6)-pyridinophane ligands RN4 (R = H, Me, iBu, secBu, neoPent, iPr, Ts) were investigated in detail. Steric properties of the alkyl group at the axial amine in the RN4 ligand were found to strongly affect the conformational preferences and dynamic behavior in solution. Several types of conformational exchange processes were revealed by variable-temperature NMR and 2D exchange spectroscopy, including degenerative exchange in a pseudotetrahedral species as well as exchange between two isomers with different conformers of tri- and tetracoordinate RN4 ligands. These exchange processes are slower for the complexes containing bulky alkyl groups at the amine compared to less sterically demanding analogues. A clear correlation is also observed between the steric bulk of the alkyl substituents and the photoluminescent properties of the derived complexes, with less dynamic complexes bearing bulkier alkyl substituents exhibiting higher absolute photoluminescence quantum yield (PLQY) in solution and the solid state: PLQY in solution increases in the order Me < neoPent < iBu < secBu ≈ iPr < tBu. The electrochemical properties of the cationic complexes [(RN4)CuI(MeCN)]X (X = BF4, PF6) were also dependent on the steric properties of the amine substituent.

Ligand-Induced Variations in Structural and Dynamical Properties Within an Enzyme Superfamily.

Enzyme catalysis is a complex process involving several steps along the reaction coordinates, including substrate recognition and binding, chemical transformation, and product release. Evidence continues to emerge linking the functional and evolutionary role of conformational exchange processes in optimal catalytic activity. Ligand binding changes the conformational landscape of enzymes, inducing long-range conformational rearrangements. Using functionally distinct members of the pancreatic ribonuclease superfamily as a model system, we characterized the structural and conformational changes associated with the binding of two mononucleotide ligands. By combining NMR chemical shift titration experiments with the chemical shift projection analysis (CHESPA) and relaxation dispersion experiments, we show that biologically distinct members of the RNase superfamily display discrete chemical shift perturbations upon ligand binding that are not conserved even in structurally related members. Amino acid networks exhibiting coordinated chemical shift displacements upon binding of the two ligands are unique to each of the RNases analyzed. Our results reveal the contribution of conformational rearrangements to the observed chemical shift perturbations. These observations provide important insights into the contribution of the different ligand binding specificities and effects of conformational exchange on the observed perturbations associated with ligand binding for functionally diverse members of the pancreatic RNase superfamily.

Residue selective 15N CEST and CPMG experiments for studies of millisecond timescale protein dynamics

Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes on the millisecond timescale. Traditional pseudo 3D 15N CEST and CPMG experiments have certain limitations in their applications. For example, both experiments have low sensitivity for broadened resonances, and the process of optimizing sample conditions and experimental parameters are often time consuming. To overcome these limitations, we herein present a new set of residue selective 15N CEST and CPMG pulse sequences by employing the Hartmann-Hahn cross-polarization transfer of magnetization in both 1D and 2D schemes. Combined with frequency labeling in the indirect dimension using only a small number of increments, the pulse sequences in the 2D scheme can be applied on resonances in overlapped regions of the 1H-15N HSQC spectrum. The pulse sequences were further applied on several proteins, demonstrating their advantages over the traditional CEST and CPMG experiments under specific circumstances.

Ultrafast Through-Space Electronic Energy Transfer in Molecular Dyads Built around Dynamic Spacer Units.

A pair of complementary molecular dyads have been synthesized around a 1,2-diaminocyclohexyl spacer that itself undergoes ring inversion. Despite these conformational exchange processes, the donor and acceptor occupy quite restricted spatial regions, and they are not interchangeable. The donor and acceptor pair comprise disparate boron dipyrromethene dyes selected to display favorable electronic energy transfer (EET). Steady-state emission spectroscopy confirms that through-space EET from donor to acceptor is almost quantitative, aided by the relatively short separations. Ultrafast time-resolved fluorescence spectroscopy has allowed determination of the rates of EET for both dyads. Surprisingly, in view of the close proximity of donor and acceptor (center-to-center separations less than 20 Å), the EET dynamics are well-accounted for in terms of the computed molecular conformations and conventional Förster theory. One dyad appears as a single family of conformations, but EET for the second dyad corresponds to dual-exponential kinetics. In this latter case, an intramolecular hydrogen bond helps stabilize an open geometry, wherein EET is relatively slow.

Palladium Complexes of N,N'-Bis(2-aminoethyl)oxamide (H2L): Structural (PdIIL, PdII2L2, and PdIVLCl2), Electrochemical, Dynamic 1H NMR, and Cytotoxicity Studies.

The monomeric (PdL·2H2O) and dimeric (Pd2L2·7H2O) palladium(II) complexes of N,N'-bis(2-aminoethyl)oxamide (H2L) were isolated, and their structures were established by single-crystal X-ray diffraction. Both compounds display identical cis-(2Namide + 2Namine) coordination environments of the metal ion. The dimer, representing a combination of two PdL species with an open lateral chelate ring, has an "open clamshell"-like structure. The intramolecular metal-metal separation in Pd2L2 (3.215 Å) is slightly shorter than the sum of the van der Waals radii of the palladium(II) atoms. The dimeric complex is relatively stable to dissociation, and its spectral features in aqueous solutions have been compared to those of the monomeric complex. A 1H NMR spectroscopic study revealed the presence of the dynamic conformational exchange process assigned to a turning of the dimeric molecule "inside out" with an activation energy of 65 kJ/mol. Cyclic voltammetry of PdL in perchlorate-, chloride-, and sulfate-containing electrolytes revealed two-electron oxidation of the palladium center. For the dimeric complex similar, though irreversible, oxidation to the palladium(IV) state was observed in NaCl electrolyte. At the same time, in NaClO4 or Na2SO4 solutions oxidation of Pd2L2 occurs in two distinct steps. The first step is quasi-reversible and can be assigned to the formation of species in an intermediate PdIIIPdIII state. Monomeric palladium(IV) complex PdIVLCl2 was generated via chemical oxidation of PdIIL by peroxodisulfate in the presence of chloride ions and structurally characterized. The related MIIL complexes (M = Pd, Ni, Cu) showed low cytotoxicity in human cancer cell lines AGS (gastric adenocarcinoma) and HCT116 (colorectal carcinoma) with IC50 values from 204 to 525 μM, while the proligand H2L was devoid of antiproliferative activity (IC50 > 1000 μM).

NMR experiments for the rapid identification of P=O···H-X type hydrogen bonds in nucleic acids.

Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927-7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1H,31P-correlation experiments as well as a BEST-HNP experiment exploiting 3hJN,P rather than 2hJH,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~ 100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2'-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.

Practical considerations for investigation of protein conformational dynamics by 15N R 1ρ relaxation dispersion.

It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure ("excited states") has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by 15N R 1ρ relaxation dispersion. The schemes use selective Hartmann-Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713-721, 2005; Hansen et al. in J Am Chem Soc 131:3818-3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R 1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.