Transient Dimer Research Articles

Photochemical Origins of Iron Flocculation in Acid Mine Drainage.

Acid mine drainage (AMD) raises a global environmental concern impacting the iron cycle. Although the formation of Fe(III) minerals in AMD-impacted waters has previously been reported to be regulated by biological processes, the role of abiotic processes remains largely unknown. This study first reported that a photochemical reaction coupled with O2 significantly accelerated the formation of Fe(III) flocculates (i.e., schwertmannite) in the AMD, as evidenced by the comparison of samples from contaminated sites across different natural conditions at latitudes 24-29° N. Combined with experimental and modeling results, it is further discovered that the intramolecular oxidation of photogenerated Fe(II) with a five-coordinative pyramidal configuration (i.e., [(H2O)5Fe]2+) by O2 was the key in enhancing the photooxidation of Fe(II) in the simulated AMD. The in situ attenuated total reflectance-Fourier transform infrared spectrometry (ATR-FTIR), UV-vis spectroscopy, solvent substitution, and quantum yield analyses indicated that, acting as a precursor for flocculation, [(H2O)5Fe]2+ likely originated from both the dissolved and colloidal forms of Fe(III) through homogeneous and surface ligand-to-metal charge transfers. Density functional theory calculations and X-ray absorption spectroscopy results further suggested that the specific oxidation pathways of Fe(II) produced the highly reactive iron species and triggered the hydrolysis and formation of transient dihydroxo dimers. The proposed new pathways of Fe cycle are crucial in controlling the mobility of heavy metal anions in acidic waters and enhance the understanding of complicated iron biochemistry that is related to the fate of contaminants and nutrients.

Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease

N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 310-helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.

Perylene-derivative singlet exciton fission in water solution.

We provide direct evidence of singlet fission occurring with water-soluble compounds. We show that perylene-3,4,9,10-tetracarboxylate forms dynamic dimers in aqueous solution, with lifetimes long enough to allow intermolecular processes such as singlet fission. As these are transient dimers rather than stable aggregates, they retain a significant degree of disorder. We performed a comprehensive analysis of such dynamic assemblies using time-resolved absorption and fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, and theoretical modelling, allowing us to observe the characteristic signatures of singlet fission and develop a model to characterize the different species observed. Our findings reveal that structure fluctuations within perylene-3,4,9,10-tetracarboxylate associations are key in favoring either singlet fission or charge separation. The efficiency of triplet formation is higher than 100%, and the disordered system leads to triplets living in the nanosecond time range.

Rotation of the c-Ring Promotes the Curvature Sorting of Monomeric ATP Synthases.

ATP synthases are proteins that catalyse the formation of ATP through the rotatory movement of their membrane-spanning subunit. In mitochondria, ATP synthases are found to arrange as dimers at the high-curved edges of cristae. Here, a direct link is explored between the rotatory movement of ATP synthases and their preference for curved membranes. An active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles is found. Coarse-grained simulations confirm the curvature-seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein-protein contacts. The formation of transient protein dimers relies on the membrane-mediated attractive interaction of the order of 1.5kB T produced by a hydrophobic mismatch upon protein rotation. Transient dimers are sustained by a conic-like arrangement characterized by a wedge angle of θ ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. The results suggest a new role of the rotational movement of ATP synthases for their dynamic self-assembly in biological membranes.

Prediction of Cu Zeolite NH3-SCR Activity from Variable Temperature 1H NMR Spectroscopy.

Selective catalytic reduction (SCR) of NOx by ammonia is one of the dominant pollution abatement technologies for near-zero NOx emission diesel engines. A crucial step in the reduction of NOx to N2 with Cu zeolite NH3-SCR catalysts is the generation of a multi-electron donating active site, implying the permanent or transient dimerization of Cu ions. Cu atom mobility has been implicated by computational chemistry as a key factor in this process. This report demonstrates how variable temperature 1H NMR reveals the Cu induced generation of sharp 1H resonances associated with a low concentration of sites on the zeolite. The onset temperature of the appearance of these signals was found to strongly correlate with the NH3-SCR activity and was observed for a range of catalysts covering multiple frameworks (CHA, AEI, AFX, ERI, ERI-CHA, ERI-OFF, *BEA), with different Si/Al ratios and different Cu contents. The results point towards universal applicability of variable temperature NMR to predict the activity of a Cu-zeolite SCR catalyst. The unique relationship of a spectroscopic feature with catalytic behavior for zeolites with different structures and chemical compositions is exceptional in heterogeneous catalysis.

A critical evaluation of protein kinase regulation by activation loop autophosphorylation.

Phosphorylation of proteins is a ubiquitous mechanism of regulating their function, localization, or activity. Protein kinases, enzymes that use ATP to phosphorylate protein substrates are, therefore, powerful signal transducers in eukaryotic cells. The mechanism of phosphoryl-transfer is universally conserved among protein kinases, which necessitates the tight regulation of kinase activity for the orchestration of cellular processes with high spatial and temporal fidelity. In response to a stimulus, many kinases enhance their own activity by autophosphorylating a conserved amino acid in their activation loop, but precisely how this reaction is performed is controversial. Classically, kinases that autophosphorylate their activation loop are thought to perform the reaction in trans, mediated by transient dimerization of their kinase domains. However, motivated by the recently discovered regulation mechanism of activation loop cis-autophosphorylation by a kinase that is autoinhibited in trans, we here review the various mechanisms of autoregulation that have been proposed. We provide a framework for critically evaluating biochemical, kinetic, and structural evidence for protein kinase dimerization and autophosphorylation, and share some thoughts on the implications of these mechanisms within physiological signaling networks.

The Self-Association of the KRAS4b Protein is Altered by Lipid-Bilayer Composition and Electrostatics.

KRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self-association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self-assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self-association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the "α4/5-α4/5" interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge-reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane.

The Self‐Association of the KRAS4b Protein is Altered by Lipid‐Bilayer Composition and Electrostatics

AbstractKRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self‐association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self‐assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self‐association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the “α4/5‐α4/5” interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge‐reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane.

PKD autoinhibition in trans regulates activation loop autophosphorylation in cis

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans, whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans-autoinhibition, followed by activation loop autophosphorylation in cis. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis. Phosphorylation serves to increase PKD activity and prevent trans-autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1

Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.

Dimers of D76N-β2-microglobulin display potent antiamyloid aggregation activity

Self-association of WT β2-microglobulin (WT-β2m) into amyloid fibrils is associated with the disorder dialysis related amyloidosis. In the familial variant D76N-β2m, the single amino acid substitution enhances the aggregation propensity of the protein dramatically and gives rise to a disorder that is independent of renal dysfunction. Numerous biophysical and structural studies on WT- and D76N-β2m have been performed in order to better understand the structure and dynamics of the native proteins and their different potentials to aggregate into amyloid. However, the structural properties of transient D76N-β2m oligomers and their role(s) in assembly remained uncharted. Here, we have utilized NMR methods, combined with photo-induced crosslinking, to detect, trap, and structurally characterize transient dimers of D76N-β2m. We show that the crosslinked D76N-β2m dimers have different structures from those previously characterized for the on-pathway dimers of ΔN6-β2m and are unable to assemble into amyloid. Instead, the crosslinked D76N-β2m dimers are potent inhibitors of amyloid formation, preventing primary nucleation and elongation/secondary nucleation when added in substoichiometric amounts with D76N-β2m monomers. The results highlight the specificity of early protein-protein interactions in amyloid formation and show how mapping these interfaces can inform new strategies to inhibit amyloid assembly.

Dimerization of β2-adrenergic receptor is responsible for the constitutive activity subjected to inverse agonism

Dimerization of beta 2-adrenergic receptor (β2-AR) has been observed across various physiologies. However, the function of dimeric β2-AR is still elusive. Here, we revealed that dimerization of β2-AR is responsible for the constitutive activity of β2-AR generating inverse agonism. Using a co-immunoimmobilization assay, we found that transient β2-AR dimers exist in a resting state, and the dimer was disrupted by the inverse agonists. A Gαs preferentially interacts with dimeric β2-AR, but not monomeric β2-AR, in a resting state, resulting in the production of a resting cAMP level. The formation of β2-AR dimers requires cholesterol on the plasma membrane. The cholesterol did not interfere with the agonist-induced activation of monomeric β2-AR, unlike the inverse agonists, implying that the cholesterol is a specific factor regulating the dimerization of β2-AR. Our model not only shows the function of dimeric β2-AR but also provides a molecular insight into the mechanism of the inverse agonism of β2-AR.

Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids.

The mechanistic details behind the activation of lecithin-cholesterol acyltransferase (LCAT) by apolipoprotein A-I (apoA-I) and its mimetic peptides are still enigmatic. Resolving the fundamental principles behind LCAT activation will facilitate the design of advanced HDL-mimetic therapeutic nanodiscs for LCAT deficiencies and coronary heart disease and for several targeted drug delivery applications. Here, we have combined coarse-grained molecular dynamics simulations with complementary experiments to gain mechanistic insight into how apoA-Imimetic peptide 22A and its variants tune LCAT activity in peptide-lipid nanodiscs. Our results highlight that peptide 22A forms transient antiparallel dimers in the rim of nanodiscs. The dimerization tendency considerably decreases with the removal of C-terminal lysine K22, which has also been shown to reduce the cholesterol esterification activity of LCAT. In addition, our simulations revealed that LCAT prefers to localize to the rim of nanodiscs in a manner that shields the membrane-binding domain (MBD), αA-αA', and the lid amino acids from the water phase, following previous experimental evidence. Meanwhile, the location and conformation of LCAT in the rim of nanodiscs are spatially more restricted when the active site covering the lid of LCAT is in the open form. The average location and spatial dimensions of LCAT in its open form were highly compatible with the electron microscopy images. All peptide 22A variants studied here had a specific interaction site in the open LCAT structure flanked by the lid and MBD domain. The bound peptides showed different tendencies to form antiparallel dimers and, interestingly, the temporal binding site occupancies of the peptide variants affected their in vitro ability to promote LCAT-mediated cholesterol esterification.

Simulations of sphere-forming diblock copolymer melts

Molecular dynamics simulations are used to study melts of asymmetric sphere-forming diblock copolymers with two significantly different values of the invariant degree of polymerization, $\overline{N}=$ 3820 and 960. In both systems, changes in parameters that correspond to decreasing temperature lead to the appearance of micelles at a critical micelle temperature (CMT) and crystallization at a lower order-disorder transition temperature (ODT). The CMT is identifiable in simulations by the appearance of large clusters with a strongly segregated core region, but has no equally clear signature in scattering experiments on systems of modest $\overline{N}$. The value of the product $\ensuremath{\chi}N$ at the CMT (where $\ensuremath{\chi}$ is the Flory-Huggins parameter and $N$ is degree of polymerization) is close to that predicted by SCFT for the ODT, while the value at the actual ODT is larger and increases with decreasing $\overline{N}$. Micelles exhibit significant and comparable dispersity in aggregation number in the crystalline and liquid phases near the ODT. Both the liquid and crystal phases exhibit transient dimers consisting of pairs of neighboring spherical micelles with cores connected by a bridge of core-block material.

Microsecond MD Simulations of the Plexin-B1 RBD: N-H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry.

Amide-bond equilibrium probability density, Peq = exp(-u) (u, local potential), and associated conformational entropy, Sk = -∫Peq (ln Peq) dΩ ─ln ∫dΩ, are derived for the Rho GTPase binding domain of Plexin-B1 (RBD) as monomer and dimer from 1 μs MD simulations. The objective is to elucidate the effect of dimerization on the dynamic structure of the RBD. Dispersed (peaked) Peq functions indicate "flexibility" ("rigidity"; the respective concepts are used below in this context). The L1 and L3 loops are throughout highly flexible, the L2 loop and the secondary structure elements are generally rigid, and the L4 loop is flexible in the monomer and rigid in the dimer. Overall, many residues are more flexible in the dimer. These features, and their implications, are discussed. Unexpectedly, we find that monomer unit 1 of the dimer (in short, d1) is unusually flexible, whereas monomer unit 2 (in short, d2) is as rigid as the RBD monomer. This is revealed due to their engagement in slow-to-intermediate conformational exchange detected previously by 15N relaxation experiments. Such motions occur with rates on the order of 103-104 s-1; hence, they cannot be completely sampled over the course of 1 μs simulation. However, the extent to which rigid d2 is affected is small enough to enable physically relevant analysis. The entropy difference between d2 and the monomer yields an entropic contribution of -7 ± 0.7 kJ/mol to the free energy of RBD dimerization. In previous work aimed at similar objectives we used 50-100 ns MD simulations. Those results and the present result differ considerably. In summary, bond-vector Peq functions derived directly from long MD simulations are useful descriptors of protein structural dynamics and provide accurate conformational entropy. Within the scope of slow conformational exchange, they can be useful, even in the presence of incomplete sampling.

ZmLBD5 Increases Drought Sensitivity by Suppressing ROS Accumulation in Arabidopsis.

Drought stress is known to significantly limit crop growth and productivity. Lateral organ boundary domain (LBD) transcription factors—particularly class-I members—play essential roles in plant development and biotic stress. However, little information is available on class-II LBD genes related to abiotic stress in maize. Here, we cloned a maize class-II LBD transcription factor, ZmLBD5, and identified its function in drought stress. Transient expression, transactivation, and dimerization assays demonstrated that ZmLBD5 was localized in the nucleus, without transactivation, and could form a homodimer or heterodimer. Promoter analysis demonstrated that multiple drought-stress-related and ABA response cis-acting elements are present in the promoter region of ZmLBD5. Overexpression of ZmLBD5 in Arabidopsis promotes plant growth under normal conditions, and suppresses drought tolerance under drought conditions. Furthermore, the overexpression of ZmLBD5 increased the water loss rate, stomatal number, and stomatal apertures. DAB and NBT staining demonstrated that the reactive oxygen species (ROS) decreased in ZmLBD5-overexpressed Arabidopsis. A physiological index assay also revealed that SOD and POD activities in ZmLBD5-overexpressed Arabidopsis were higher than those in wild-type Arabidopsis. These results revealed the role of ZmLBD5 in drought stress by regulating ROS levels.

Unidirectional mannitol synthesis of Acinetobacter baumannii MtlD is facilitated by the helix–loop–helix-mediated dimer formation

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix–loop–helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.

Gated Transient Dissipative Dimerization of DNA Tetrahedra Nanostructures for Programmed DNAzymes Catalysis.

Transient dissipative dimerization and transient gated dimerization of DNA tetrahedra nanostructures are introduced as functional modules to emulate transient and gated protein–protein interactions and emergent protein–protein guided transient catalytic functions, operating in nature. Four tetrahedra are engineered to yield functional modules that, in the presence of pre-engineered auxiliary nucleic acids and the nicking enzyme Nt.BbvCI, lead to the fueled transient dimerization of two pairs of tetrahedra. The dynamic transient formation and depletion of DNA tetrahedra are followed by transient FRET signals generated by fluorophore-labeled tetrahedra. The integration of two inhibitors within the mixture of the four tetrahedra and two auxiliary modules, fueling the transient dimerization, results in selective inhibitor-guided gated transient dimerization of two different DNA tetrahedra dimers. Kinetic models for the dynamic transient dimerization and gated transient dimerization of the DNA tetrahedra are formulated and computationally simulated. The derived rate-constants allow the prediction and subsequent experimental validation of the performance of the systems under different auxiliary conditions. In addition, by appropriate modification of the four tetrahedra structures, the triggered gated emergence of selective transient catalytic functions driven by the two pairs of DNA tetrahedra dimers is demonstrated.

Molecular dynamics simulation, DFT calculations and vibrational spectroscopic study of N[sbnd]H•••O bound dimer models for DL-β-phenylalanine and 3-amino-3-(4-chlorophenyl)propionic acid

Zwitterionic NH•••O bonded dimer structures have been proposed one each for DL-β-Phenylalanine and 3-Amino-3-(4-chlorophenyl)propionic acid. Stable zwitterionic monomer possessing NH3+ and COO¯ moieties in implicit aqueous solvation were computed at B3LYP/6-311++G** level and used for classical MD simulations. MD simulations yielded, among many NH•••O bonded transient dimer and trimer species, the two dimer structures with longest lifetime τ in water medium (τ = 111.67 ps for DL-β-Phenylalanine and 111.53 ps for 3-Amino-3-(4-chlorophenyl)propionic acid). The first peaks in radial distribution functions (RDF) confirmed the formation of NH•••O bonded dimer structures, one each for DL-β-Phenylalanine and 3-Amino-3-(4-chlorophenyl)propionic acid; the first minima determined the cut-off values for the inter-molecular H•••O distances. Further, the first minimum-solvation shell also indicated one zwitterionic dimer structure with 10 water molecules as solvent medium. Experimental IR frequencies due to the NH3+ and COO¯ moieties agree within 5% of the computed values for the dimer structures. Low-frequency Raman bands near 100–50 cm−1 are assigned to the torsional vibration modes of zwitterionic NH•••O bonded dimer structures. On the role of Chlorine with respect to all the molecular properties of 3-Amino-3-(4-chlorophenyl)propionic acid vs DL-β-Phenylalanine computed from NBO, AIM and NCI calculations, its influence is not substantial but discernible enough for gaining insights into the two NH•••O bonded zwitterionic dimer species.

Stopped-flow-time-resolved SAXS for studies of ligand-driven protein dimerization.

Protein function is highly dependent on conformational changes and association or dissociation into numerous oligomeric states. Stopped-flow approaches are suitable for probing transient kinetics in proteins, and combining this approach with small-angle X-ray scattering offers an excellent probe into the structural kinetics of protein function. In this chapter we describe in detail the methodological aspects of our recent investigation of ATP-driven dimerization of nucleotide-binding domains from the bacterial transporter MsbA using stopped-flow small-angle X-ray scattering experiments. Despite extensive studies into the structure and function of MsbA, the structural-temporal insights into the conformational rearrangements and transient intermediates along the pathway in this transporter are missing. In our stopped-flow experiments we observe the rapid formation of a transient protein dimer and subsequent dimer decay over hundreds of seconds. Thus, this approach can be used to detect kinetic parameters associated with conformational changes over a wide range of time-scales for soluble and membrane proteins.