Probing Microsecond Time Scale Dynamics in Proteins by Methyl 1H Carr−Purcell−Meiboom−Gill Relaxation Dispersion NMR Measurements. Application to Activation of the Signaling Protein NtrCr

To study microsecond processes by relaxation dispersion NMR spectroscopy, low power deposition and short pulses are crucial and encourage the development of experiments that employ H-1 Carr-Purcell-Meiboom-Gill (CPMG) pulse trains. Herein, a method is described for the comprehensive study of microsecond to millisecond time scale dynamics of methyl groups in proteins, exploiting their high abundance and favorable relaxation properties. In our approach, protein samples are produced using [H-1, C-13]-D-glucose in similar to 100% D2O, which yields CHD2 methyl groups for alanine, valine, threonine, isoleucine, leucine, and methionine residues with high abundance, in an otherwise largely deuterated background. Methyl groups in such samples can be sequence-specifically assigned to near completion, using C-13 TOCSY NMR spectroscopy, as was recently demonstrated (Often, R.; et al. J. Am. Chem. Soc. 2010, 132, 2952-2960). In this Article, NMR pulse schemes are presented to measure H-1 CPMG relaxation dispersion profiles for CHD2 methyl groups, in a vein similar to that of backbone relaxation experiments. Because of the high deuteration level of methyl-bearing side chains, artifacts arising from proton scalar coupling during the CPMG pulse train are negligible, with the exception of Ile-delta 1 and Thr-gamma 2 methyl groups, and a pulse scheme is described to remove the artifacts for those residues. Strong C-13 scalar coupling effects, observed for several leucine residues, are removed by alternative biochemical and NMR approaches. The methodology is applied to the transcriptional activator NtrC(r), for which an inactive/active state transition was previously measured and the motions in the microsecond time range were estimated through a combination of backbone N-15 CPMG dispersion NMR spectroscopy and a collection of experiments to determine the exchange-free component to the transverse relaxation rate. Exchange contributions to the H-1 line width were detected for 21 methyl groups, and these probes were found to collectively report on a local structural rearrangement around the phosphorylation site, with a rate constant of (15.5 +/- 0.5) x 10(3) per second (i.e., tau(ex) = 64.7 +/- 1.9 mu s). The affected methyl groups indicate that, already before phosphorylation, a substantial, transient rearrangement takes place between helices 3 and 4 and strands 4 and 5. This conformational equilibrium allows the protein to gain access to the active, signaling state in the absence of covalent modification through a shift in a pre-existing dynamic equilibrium. Moreover, the conformational switching maps exactly to the regions that differ between the solution NMR structures of the fully inactive and active states. These results demonstrate that a cost-effective and quantitative study of protein methyl group dynamics by H-1 CPMG relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone N-15 relaxation dispersion NMR. The use of methyl groups as dynamics probes extends such applications also to larger proteins

Optimisation of NMR dynamic models I. Minimisation algorithms and their performance within the model-free and Brownian rotational diffusion spaces

The key to obtaining the model-free description of the dynamics of a macromolecule is the optimisation of the model-free and Brownian rotational diffusion parameters using the collected R1, R2 and steady-state NOE relaxation data. The problem of optimising the chi-squared value is often assumed to be trivial, however, the long chain of dependencies required for its calculation complicates the model-free chi-squared space. Convolutions are induced by the Lorentzian form of the spectral density functions, the linear recombinations of certain spectral density values to obtain the relaxation rates, the calculation of the NOE using the ratio of two of these rates, and finally the quadratic form of the chi-squared equation itself. Two major topological features of the model-free space complicate optimisation. The first is a long, shallow valley which commences at infinite correlation times and gradually approaches the minimum. The most severe convolution occurs for motions on two timescales in which the minimum is often located at the end of a long, deep, curved tunnel or multidimensional valley through the space. A large number of optimisation algorithms will be investigated and their performance compared to determine which techniques are suitable for use in model-free analysis. Local optimisation algorithms will be shown to be sufficient for minimisation not only within the model-free space but also for the minimisation of the Brownian rotational diffusion tensor. In addition the performance of the programs Modelfree and Dasha are investigated. A number of model-free optimisation failures were identified: the inability to slide along the limits, the singular matrix failure of the Levenberg–Marquardt minimisation algorithm, the low precision of both programs, and a bug in Modelfree. Significantly, the singular matrix failure of the Levenberg–Marquardt algorithm occurs when internal correlation times are undefined and is greatly amplified in model-free analysis by both the grid search and constraint algorithms. The program relax (http://www.nmr-relax.com) is also presented as a new software package designed for the analysis of macromolecular dynamics through the use of NMR relaxation data and which alleviates all of the problems inherent within model-free analysis

Temperature Dependence of Backbone Dynamics in Human Ileal Bile Acid-Binding Protein: Implications for the Mechanism of Ligand Binding

Human ileal bile acid-binding protein (I-BABP), a member of the family of intracellular lipid binding proteins plays a key role in the cellular trafficking and metabolic regulation of bile salts. The protein has two internal and, according to a recent study, an additional superficial binding site and binds di- and trihydroxy bile salts with positive cooperativity and a high degree of site-selectivity. Previously, in the apo form, we have identified an extensive network of conformational fluctuations on the millisecond time scale, which cease upon ligation. Additionally, ligand binding at room temperature was found to be accompanied by a slight rigidification of picosecond-nanosecond (ps-ns) backbone flexibility. In the current study, temperature-dependent N-15 NMR spin relaxation measurements were used to gain more insight into the role of dynamics in human I-BABP-bile salt recognition. According to our analysis, residues sensing a conformational exchange in the apo state can be grouped into two clusters with slightly different exchange rates. The entropy-enthalpy compensation observed for both clusters suggests a disorder-order transition between a ground and a sparsely populated higher energy state in the absence of ligands. Analysis of the faster, ps-ns motion of N-15-H-1 bond vectors indicates an unusual nonlinear temperature-dependence for both ligation states. Intriguingly, while bile salt binding results in a more uniform response to temperature change throughout the protein, the temperature derivative of the generalized order parameter shows different responses to temperature increase for the two forms of the protein in the investigated temperature range. Analysis of both slow and fast motions in human I-BABP indicates largely different energy landscapes for the apo and halo states suggesting that optimization of binding interactions might be achieved by altering the dynamic behavior of specific segments in the protein

Protein Structure along the Order–Disorder Continuum

Thermal fluctuations cause proteins to adopt an ensemble of conformations wherein the relative stability of the different ensemble members is determined by the topography of the underlying energy landscape. “Folded” proteins have relatively homogeneous ensembles, while “unfolded” proteins have heterogeneous ensembles. Hence, the labels “folded” and “unfolded” represent attempts to provide a qualitative characterization of the extent of structural heterogeneity within the underlying ensemble. In this work, we introduce an information-theoretic order parameter to quantify this conformational heterogeneity. We demonstrate that this order parameter can be estimated in a straightforward manner from an ensemble and is applicable to both unfolded and folded proteins. In addition, a simple formula for approximating the order parameter directly from crystallographic B factors is presented. By applying these metrics to a large sample of proteins, we show that proteins span the full range of the order–disorder axis.National Institutes of Health (U.S.) (NIH Grant 5R21NS063185-02

Determination of Conformational Equilibria in Proteins Using Residual Dipolar Couplings

In order to carry out their functions, proteins often undergo significant conformational fluctuations that enable them to interact with their partners. The accurate characterization of these motions is key in order to understand the mechanisms by which macromolecular recognition events take place. Nuclear magnetic resonance spectroscopy offers a variety of powerful methods to achieve this result. We discuss a method of using residual dipolar couplings as replica-averaged restraints in molecular dynamics simulations to determine large amplitude motions of proteins, including those involved in the conformational equilibria that are established through interconversions between different states. By applying this method to ribonuclease A, we show that it enables one to characterize the ample fluctuations in interdomain orientations expected to play an important functional role

Insights into Protein Aggregation by NMR Characterization of Insoluble SH3 Mutants Solubilized in Salt-Free Water

Protein aggregation in vivo has been extensively associated with a large spectrum of human diseases. On the other hand, mechanistic insights into protein aggregation in vitro were incomplete due to the inability in solubilizing insoluble proteins for high-resolution biophysical investigations. However, a new avenue may be opened up by our recent discovery that previously-thought insoluble proteins can in fact be solubilized in salt-free water. Here we use this approach to study the NMR structural and dynamic properties of an insoluble SH3 mutant with a naturally-occurring insertion of Val22 at the tip of the diverging turn. The obtained results reveal: 1) regardless of whether the residue is Val, Ala, Asp or Arg, the insertion will render the first hNck2 SH3 domain to be insoluble in buffers. Nevertheless, all four mutants could be solubilized in salt-free water and appear to be largely unfolded as evident from their CD and NMR HSQC spectra. 2) Comparison of the chemical shift deviations reveals that while in V22-SH3 the second helical region is similarly populated as in the wild-type SH3 at pH 2.0, the first helical region is largely unformed. 3) In V22-SH3, many non-native medium-range NOEs manifest to define non-native helical conformations. In the meanwhile a small group of native-like long-range NOEs still persists, indicating the existence of a rudimentary native-like tertiary topology. 4) Although overall, V22-SH3 has significantly increased backbone motions on the ps-ns time scale, some regions still own restricted backbone motions as revealed by analyzing 15N relaxation data. Our study not only leads to the establishment of the first high-resolution structural and dynamic picture for an insoluble protein, but also shed more light on the molecular events for the nonhierarchical folding mechanism. Furthermore, a general mechanism is also proposed for in vivo protein aggregation triggered by the genetic mutation and posttranslational modification

VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn2+ and Zn2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies

Accessing ns–μs side chain dynamics in ubiquitin with methyl RDCs

This study presents the first application of the model-free analysis (MFA) (Meiler in J Am Chem Soc 123:6098–6107, 2001; Lakomek in J Biomol NMR 34:101–115, 2006) to methyl group RDCs measured in 13 different alignment media in order to describe their supra-τc dynamics in ubiquitin. Our results indicate that methyl groups vary from rigid to very mobile with good correlation to residue type, distance to backbone and solvent exposure, and that considerable additional dynamics are effective at rates slower than the correlation time τc. In fact, the average amplitude of motion expressed in terms of order parameters S2 associated with the supra-τc window brings evidence to the existence of fluctuations contributing as much additional mobility as those already present in the faster ps-ns time scale measured from relaxation data. Comparison to previous results on ubiquitin demonstrates that the RDC-derived order parameters are dominated both by rotameric interconversions and faster libration-type motions around equilibrium positions. They match best with those derived from a combined J-coupling and residual dipolar coupling approach (Chou in J Am Chem Soc 125:8959–8966, 2003) taking backbone motion into account. In order to appreciate the dynamic scale of side chains over the entire protein, the methyl group order parameters are compared to existing dynamic ensembles of ubiquitin. Of those recently published, the broadest one, namely the EROS ensemble (Lange in Science 320:1471–1475, 2008), fits the collection of methyl group order parameters presented here best. Last, we used the MFA-derived averaged spherical harmonics to perform highly-parameterized rotameric searches of the side chains conformation and find expanded rotamer distributions with excellent fit to our data. These rotamer distributions suggest the presence of concerted motions along the side chains

Structural Biology by NMR: Structure, Dynamics, and Interactions

The function of bio-macromolecules is determined by both their 3D structure and conformational dynamics. These molecules are inherently flexible systems displaying a broad range of dynamics on time-scales from picoseconds to seconds. Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as the method of choice for studying both protein structure and dynamics in solution. Typically, NMR experiments are sensitive both to structural features and to dynamics, and hence the measured data contain information on both. Despite major progress in both experimental approaches and computational methods, obtaining a consistent view of structure and dynamics from experimental NMR data remains a challenge. Molecular dynamics simulations have emerged as an indispensable tool in the analysis of NMR data