Closed-cleft Conformation Research Articles

Conformational Free Energies of Metabotropic Glutamate Receptor Ligand-Binding Domains

Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that mediate cellular responses to the neurotransmitter glutamate in the central nervous system. Crystal structures and single-molecule FRET results suggest that closure of the ligand-binding domains (LBDs) as well as reorientation of the dimer interface are necessary conformational transitions for receptor activation. The energetic factors that drive these coupled processes, however, are poorly understood. Here, we report free energy landscapes, or potentials of mean force (PMFs), for monomer mGluR3 LBDs in apo and glutamate-bound states as well as dimer mGluR3 LBDs the active and inactive poses. PMFs of LBD monomers suggest the glutamate-bound LBD is bistable: the closed and open-cleft conformations are easily accessible. In apo LBDs, the closed-cleft conformation is destabilized relative to the glutamate-bound. In contrast, PMFs for glutamate-bound dimers indicate a strong preference for the closed state in both active and inactive poses. These results suggest cooperativity between dimer subunits. Specifically, network analysis suggests subunit coupling alters the dynamics of an alpha helix in the LBD lower lobe. The energetics corresponding to the reorientation of the dimer interface are also examined.

Structural Basis of the Proton Sensitivity of Human GluN1-GluN2A NMDA Receptors

N-methyl-D-aspartate (NMDA) receptors are critical for synaptic development and plasticity. While glutamate is the primary agonist, protons can modulate NMDA receptor activity at synapses during vesicle exocytosis by mechanisms that are unknown. We used cryo-electron microscopy to solve the structures of the human GluN1-GluN2A NMDA receptor at pH 7.8 and pH 6.3. Our structures demonstrate that the proton sensor predominantly resides in the N-terminal domain (NTD) of the GluN2A subunit and reveal the allosteric coupling mechanism between the proton sensor and the channel gate. Under high-pH conditions, the GluN2A-NTD adopts an "open-and-twisted" conformation. However, upon protonation at the lower pH, the GluN2A-NTD transitsfrom an open- to closed-cleft conformation, causingrearrangements between the tetrameric NTDs andagonist-binding domains. The conformational mobility observed in our structures (presumably from protonation) is supported by molecular dynamics simulation. Our findings reveal the structural mechanisms by which protons allosterically inhibit human GluN1-GluN2A receptor activity.

High Conformational Variability in the GluK2 Kainate Receptor Ligand-Binding Domain

The kainate family of ionotropic glutamate receptors (iGluRs) mediates pre- and postsynaptic neurotransmission. Previously computed conformational potentials of mean force (PMFs) for iGluR ligand-binding domains (LBDs) revealed subtype-dependent conformational differences between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) iGluR subfamilies. Here we report PMFs for the kainate receptor GluK2 in apo and glutamate-bound states. Apo and glutamate-bound GluK2 LBDs preferentially access closed-cleft conformations.Apo GluK2 exhibits a surprisingly high degree of conformational flexibility, accessing open and closed states. Comparing across iGluR subtypes, these results are similar to glycine-binding GluN1 and GluN3A NMDA subunits and differ from glutamate-binding GluA2 and GluN2A subunits. To test the contribution of cross-lobe interactions onclosed-cleft LBD stability, we computed PMFs for twoGluK2 mutants, D462A and D656S. D462A, butnot D656S, weakens closed-cleft conformations of the glutamate-bound LBD. Theoretical Boltzmann-weighted small-angle X-ray scattering profilesimprove agreement with experimental results compared with calculations from the LBD crystal structure alone.

Free Energy Landscapes of Metabotropic Glutamate Receptor Ligand-Binding Domains

Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that mediate cellular responses to the neurotransmitter glutamate in the central nervous system. Crystal structures and single-molecule FRET results suggest that closure of the ligand-binding domains (LBDs) as well as reorientation of the dimer interface are necessary conformational transitions for receptor activation. The energetic factors that drive these coupled processes, however, are poorly understood. Here, we report free energy landscapes, or potentials of mean force (PMFs), for monomer and dimer mGluR3 LBDs in apo and glutamate-bound states. PMFs of LBD monomers suggest the LBDs are bistable: the closed and open-cleft conformations are accessible for both glutamate-bound and apo LBDs, but with the apo closed-cleft conformation destabilized relative to the glutamate-bound LBD.

Intra and Interdomain Motions of the NMDA Receptor using Single Molecule FRET

Ionotropic glutamate receptors are tetrameric ligand-gated ion channels which mediate the majority of excitatory neurotransmission in the central nervous system. The NMDA receptor (NMDAR) in particular is unique in that it is an obligate heterotetramer that also requires a co-agonist glycine or D-serine. The amino-terminal domains (ATD) and ligand-binding domains (LBD) of the NMDAR have been shown to fold into a clamshell-like shape that close upon the introduction of their respective ligand. At the LBD, extent of cleft closure of the agonist-binding domain is one mechanism by which the agonist mediates channel activity. However, only an open-cleft or a closed-cleft conformation has been seen in crystal structures of the glycine-binding GluN1 subunit of the NMDAR, and no partially-closed cleft states have been observed, leaving unanswered the mechanisms of partial agonism. Similarly, it is still unclear how binding of allosteric modulators at the NMDAR ATD is propagated downward through the LBD and transmembrane domain to affect channel activity. Here, we have used single molecule FRET to examine NMDAR dynamics in order to address these questions. Unnatural amino acids were used to achieve specific or directional labeling of the protein. Our studies in the LBD reveal experimental evidence of the two-state model, with a common closed cleft conformation among different agonists with agonist efficacy dictated by its ability to stabilize that closed cleft. We also see that this stability, as measured by the fractional occupancy of the isolated domain in cleft-closure states below a threshold, correlates linearly with agonist efficacy, providing a link between LBD dynamics, cleft closure, and channel activity. Our inter-domain studies on the full-length receptor reveal large-scale intersubunit motions and striking differences in the conformational landscape when bound to agonist and allosteric modulators.

Conformational Dynamics of the GluK2 Ligand-Binding Domain

The kainate family of ionotropic glutamate receptors (GluK) mediates neurotransmission in pre- and post-synaptic neurons. Closure of the ligand-binding domain (LBD) in response to agonist binding is a necessary conformational transition to gate the ion channel. Previously computed free energy landscapes for iGluR LBDs revealed subtype-dependent conformational differences between AMPA and NMDA iGluR families, which may help to explain functional differences between iGluR subtypes. Here we report free energy landscapes, or potentials of mean force (PMFs), for the GluK2 LBD in apo and glutamate-bound states. GluK2 PMFs reveal both apo and glutamate-bound LBDs preferentially access a closed-cleft conformation, consistent with a conformational selection mechanism for binding. These results are also consistent with PMFs for NMDA but not AMPA receptor LBDs. Additionally, the open-cleft conformation is more accessible to the GluK2 apo state than the glutamate-bound state, consistent with previous results for both NMDA and AMPA receptors. Small-angle X-ray scattering profiles for the apo- and glutamate-bound states calculated from a Boltzmann-weighted ensemble of conformations are compared with experimental SAXS profiles. Finally, structural and biochemical properties of GluK2 in its apo state are probed using proteolytic protection assays, circular dichroism, and multi-angle light scattering.

Kinetic Contributions to Gating by Interactions Unique to N-methyl-d-aspartate (NMDA) Receptors

Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses.

Structural Basis for Lack of ADP-ribosyltransferase Activity in Poly(ADP-ribose) Polymerase-13/Zinc Finger Antiviral Protein

The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD(+) binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity.

NMDA Receptor smFRET Studies Reveal Role of Dynamics of the Agonist-Binding Domain in Mediating Agonist Efficacy

Ionotropic glutamate receptors are tetrameric ligand-gated ion channels which mediate the majority of excitatory neurotransmission in the central nervous system. This family is subdivided into three classes: the AMPA receptor, the kainate receptor, and the NMDA receptor. Extent of cleft closure of the agonist-binding domain is one mechanism by which the agonist mediates channel activity for a number of the glutamate receptor subtypes. However, only an open-cleft or a closed-cleft conformation has seen in crystal structures of the glycine-binding GluN1 subunit of the NMDA receptor, and no partially-closed cleft states have been observed. Here, we have used single molecule FRET to examine the dynamics of the NMDA receptor specifically with respect to the cleft closure conformational change of the isolated agonist-binding domain of GluN1 when bound to ligands of varying efficacy. These studies reveal differences in the range of cleft closure states occupied by the agonist-binding domain with the antagonist DCKA-bound form and the full agonist glycine-bound form showing a large range of cleft closure states, while the partial agonists ACBC and L-alanine, as well as the full agonist D-serine, have a much narrower spread in their cleft closure states. Further analysis shows that the fractional occupancy of the isolated domain in cleft-closure states below a threshold does correlate with agonist efficacy, providing a link between agonist-binding domain dynamics, along with cleft closure, and channel activity.

Free Energy Landscapes for a Kainate Receptor Ligand-Binding Domain

The kainate family of ionotropic glutamate receptors (GluK) mediate neurotransmission in pre- and post-synaptic neurons. Previously computed free energy landscapes for iGluR ligand-binding domains (LBD) revealed subtype-dependent conformational differences between NMDA and AMPA iGluR families. Here we report free energy landscapes for the GluK2 ligand-binding domain (LBD) in apo and glutamate-bound states. The GluK2 free energy landscapes reveal both apo and glutamate-bound LBDs preferentially access the closed-cleft conformation and suggest a conformational selection mechanism of binding. These results are consistent with the free energy landscapes for NMDA receptors but not AMPA receptors. Additionally, the open-cleft conformation is more accessible to the GluK2 apo state than the glutamate-bound state, consistent with previous results for both NMDA and AMPA receptors. Finally, large-scale structural dynamics are characterized by principle component analysis.

Crystal structures and kinetics of Type III 3-phosphoglycerate dehydrogenase reveal catalysis by lysine.

D-Phosphoglycerate dehydrogenase (PGDH) catalyzes the first committed step of the phosphorylated serine biosynthesis pathway. Here, we report for the first time, the crystal structures of Type IIIK PGDH from Entamoeba histolytica in the apo form, as well as in complexes with substrate (3-phosphoglyceric acid) and cofactor (NAD(+) ) to 2.45, 1.8 and 2.2 Å resolution, respectively. Comparison of the apo structure with the substrate-bound structure shows that the substrate-binding domain is rotated by ~ 20° to close the active-site cleft. The cofactor-bound structure also shows a closed-cleft conformation, in which NAD(+) is bound to the nucleotide-binding domain and a formate ion occupies the substrate-binding site. Superposition of the substrate- and cofactor-bound structures represents a snapshot of the enzyme in the active form, where C2 of the substrate and C4N of the cofactor are 2.2 Å apart, and the amino group of Lys263 is close enough to the substrate to remove the proton from the hydroxyl group of PGA, indicating the role of Lys in the catalysis. Mutation of Lys263 to Ala yields just 0.8% of the specific activity of the wild-type enzyme, revealing that Lys263 indeed plays an integral role in the catalytic activity. The detectable activity of the mutant, however, indicates that after 20° rotation of the substrate-binding domain, the resulting positions of the substrate and cofactor are sufficiently close to make a productive reaction.

Understanding Nucleotide-Regulated FtsZ Filament Dynamics and the Monomer Assembly Switch with Large-Scale Atomistic Simulations

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg2+ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.

NMDA and AMPA Receptor Ligand-Binding Domains Exhibit Subtype-Specific Conformational Propensities

The NMDA receptor family of ionotropic glutamate receptors (iGluRs) is formed by assemblies of GluN1, GluN2, and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states reveal open- and closed-cleft conformations, respectively. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo-state LBDs sample both open- and closed-cleft conformations, suggesting a conformational selection mechanism for agonist binding.

Role of Cross-Cleft Contacts in NMDA Receptor Gating

In response to brief glutamate exposure, NMDA receptors produce excitatory currents that have sub-maximal amplitudes and characteristically slow kinetics. The activation sequence starts when glutamate binds to residues located on the upper lobe of extracellularly located ligand-binding domains (LBDs) and then contacts lower lobe residues to bridge the cleft between the two hinged lobes. This event stabilizes a narrow-cleft LBD conformation and may facilitate subsequent inter-lobe contacts that further stabilize the closed cleft. Agonist efficacy has been traced to the degree of agonist-induced cleft-closure and may also depend on the stability of the closed-cleft conformation. To investigate how cross-cleft contacts contribute to the amplitude and kinetics of NMDA receptor response, we examined the activation reaction of GluN1/GluN2A receptors that had single-residue substitutions at the interface between LBD lobes. We found that side-chain truncations at residues of putative contact between lobes increased glutamate efficacy through independent additive mechanisms in GluN1 and GluN2A subunits. In contrast, removing side-chain charge with isosteric substitutions at the same sites decreased glutamate efficacy. These results support the view that in GluN1/GluN2A receptors’ natural interactions between residues on opposing sides of the ligand-binding cleft encode the stability of the glutamate-bound closed-cleft conformations and limit the degree of cleft closure, thus contributing to the sub-maximal response and emblematically slow NMDA receptor deactivation after brief stimulation.

Conformational Analysis of NMDA Receptor GluN1, GluN2, and GluN3 Ligand-Binding Domains Reveals Subtype-Specific Characteristics

The NMDA receptor family of glutamate receptor ion channels is formed by obligate heteromeric assemblies of GluN1, GluN2, and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states unexpectedly reveal open- and closed-cleft conformations, respectively, with water molecules filling the binding pockets. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo-state LBDs sample closed-cleft conformations, suggesting that their agonists bind via a conformational selection mechanism. By contrast, free energy landscapes for the AMPA receptor GluA2 LBD suggest binding of glutamate via an induced-fit mechanism. Principal component analysis reveals a rich spectrum of hinge bending, rocking, twisting, and sweeping motions that are different for the GluN1, GluN2A, GluN3A, and GluA2 LBDs. This variation highlights the structural complexity of signaling by glutamate receptor ion channels.

Rebuttal from David M. MacLean

The main effects of the transmembrane AMPA receptor (AMPAR) auxiliary proteins (TARPs) on AMPAR gating are slowing of deactivation and desensitization, increasing peak Popen and speeding of recovery from desensitization (Priel et al. 2005; Tomita et al. 2005). All of these observations can be reproduced by increasing the rate of recovery from desensitization, γ, and by one of two additional means: either increasing the open rate, β (as proposed by Dr Howe, 2013), or reducing the cleft opening rate, CO, and desensitization rate, δ (as suggested by myself, MacLean, 2013). Moreover, increasing the channel opening rate or decreasing the cleft open rate both result in the reported substantial left-shift in steady-state glutamate EC50 values produced by TARPs (Priel et al. 2005; Tomita et al. 2005; Kott et al. 2007) as well as the smaller left-shift in peak EC50 values (Morimoto-Tomita et al. 2009). Therefore, such concentration–response data are not helpful in distinguishing an effect on gating transitions from an effect on pre-gating transitions such as stabilizing closed-cleft states. However, peak inhibition curves using NBQX and CNQX may provide some support for the hypothesis that TARPs affect pre-gating transitions by promoting states with closed ligand-binding domains (LBDs). The LBDs of AMPARs exist in multiple closed-cleft conformations, both when the agonist is bound and in the apo state (Landes et al. 2011). If the presence of TARPs causes AMPAR LBDs to adopt more closed conformations, then even in the apo state the binding cleft would be, on average, slightly more shut. Consequently, larger ligands such as NBQX or CNQX would have slower binding rates since there would be fewer favourable collisions between these compounds and the more closed LBDs. Such slowed binding rates of larger antagonists would result in right-shifts of peak inhibition curves and this is precisely what is observed experimentally. The peak response IC50 values obtained using rapid perfusion on outside-out patches for both CNQX and NBQX are right-shifted approximately 2-fold by stargazin (MacLean & Bowie, 2011). While this observation is consistent with TARPs stabilizing more closed LBD conformations, more compelling evidence is needed. Ultimately, to discriminate between these mechanisms, direct measurements of both the degree of LBD closure and coupling between LBD closure and channel opening will be required.

Unique Conformational Distributions for NMDA Receptor Glycine and Glutamate Ligand-Binding Domains

Ionotropic glutamate receptors (iGluRs) mediate communication at most excitatory synapses in the brain. iGluRs are organized into three major families--the AMPA, kainate, and NMDA receptors. NMDA receptors are obligate heteromeric assemblies of glycine- and glutamate-binding subunits. Unexpectedly, crystal structures of the glycine-binding GluN1 and GluN3A ligand binding domains (LBDs) in their apo states reveal open and closed cleft conformations, respectively. Computed conformational free energy landscapes also exhibit minima at both open and closed cleft conformations for apo GluN1 and GluN3A LBDs. The minimum at the closed cleft conformation is preserved for the glycine-bound LBDs. In contrast, the free energy landscapes for the NMDA and AMPA receptor glutamate-binding subunits GluN2A and GluA2 show a shift in the minimum upon glutamate binding. Principal component analysis reveals a spectrum of conformational transitions that differ for the GluN1, GluN3A, GluN2A, and GluA2 LBDs. This variation highlights the structural complexity of signaling by iGluRs.

Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation

Synaptic AMPA receptors are greatly influenced by a family of transmembrane AMPA receptor regulatory proteins (TARPs) which control trafficking, channel gating and pharmacology. The prototypical TARP, stargazin (or γ2), shifts the blocking ability of several AMPAR-selective compounds including the commonly used quinoxalinedione antagonists, CNQX and NBQX. Stargazin's effect on CNQX is particularly intriguing as it not only apparently lowers the potency of block, as with NBQX, but also renders it a partial agonist. Given this, agonist behaviour by CNQX has been speculated to account for its weaker blocking effect on AMPAR-TARP complexes. Here we show that this is not the case. The apparent effect of stargazin on CNQX antagonism can be almost entirely explained by an increase in the apparent affinity for l-glutamate (l-Glu), a full agonist and neurotransmitter at AMPAR synapses. Partial agonism at best plays a minor role but not through channel gating per se but rather because CNQX elicits AMPAR desensitization. Our study reveals that CNQX is best thought of as a non-competitive antagonist at glutamatergic synapses due to the predominance of non-equilibrium conditions. Consequently, CNQX primarily reports the proportion of AMPARs available for activation but may also impose additional block by receptor desensitization.

Structural landscape of isolated agonist-binding domains from single AMPA receptors

α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors mediate fast excitatory neurotransmission by converting chemical signals into electrical signals. Thus, it is important to understand the relationship between their chemical biology and their function. Single molecule fluorescence resonance energy transfer (smFRET) was used to examine the conformations explored by the agonist binding domain of the AMPA receptor for wild type and T686 mutant proteins. Each form of the agonist binding domain exhibited a dynamic, multi-state sequential equilibrium, which could only be identified using wavelet shrinkage, a signal processing technique that removes experimental shot-noise. These results illustrate that the extent of activation is dependent not on a rigid closed cleft, but instead on the probability that a given subunit will occupy a closed cleft conformation, which in turn is not only determined by the lowest energy state but by the range of states that the protein explores.

Wavelet Shrinkage to Resolve Single Molecule FRET Structural Landscape of the Isolated Ligand Binding Domain of the AMPA Receptor

Single molecule fluorescence resonance energy transfer (smFRET) spectroscopy has provided significant advances in our understanding of the relationship between structure and function in biological systems. Currently, simplifications must be made for experimental systems, data analysis, and theoretical modeling because biomolecules often exhibit mechanistic or conformational heterogeneity. For example, it is often necessary to treat biomolecular processes as transitions between two well-define states (e.g. folded vs. unfolded) despite clear experimental evidence or theoretical predictions to the contrary. The conformations explored by the agonist binding domain of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor represent such a system. The distribution of conformations in the raw data was so wide it was not possible to extract conformational details. By employing a model-free data analysis technique called wavelet shrinkage, it was determined that each protein form comprised multi-state, sequential equilibria. The results illustrate that the extent of activation is dependent not on a rigid closed cleft, but instead on the probability that a given subunit will occupy a closed cleft conformation, which in turn is determined by the range of states that the protein explores. Also, the results emphasize both the need for and the utility of advanced data processing techniques to quantify structure and dynamics in heterogeneous systems.