Kinetic Mechanism of Conformational Switch between Bistable RNA Hairpins

Transitions between the different conformational states play a critical role in many RNA catalytic and regulatory functions. In this study, we use the Kinetic Monte Carlo method to investigate the kinetic mechanism for the conformational switches between bistable RNA hairpins. We find three types of conformational switch pathways for RNA hairpins: refolding after complete unfolding, folding through basepair-exchange pathways and through pseudoknot-assisted pathways, respectively. The result of the competition between the three types of pathways depends mainly on the location of the rate-limiting base stacks (such as the GC base stacks) in the structures. Depending on the structural relationships between the two bistable hairpins, the conformational switch can follow single or multiple dominant pathways. The predicted folding pathways are supported by the activation energy results derived from the Arrhenius plot as well as the NMR spectroscopy data.     

Transient enzyme-substrate recognition monitored by real-time NMR

Slow protein folding processes during which kinetic folding intermediates occur for an extended time can lead to aggregation and dysfunction in living cells. Therefore, protein folding helpers have evolved, which prevent proteins from aggregation and/or speed up folding processes. In this study, we present the structural characterization of a long-living transient folding intermediate of RNase T1 (S54G/P55N) by time-resolved NMR spectroscopy. NMR resonances of this kinetic folding intermediate could be assigned mainly by a real-time 3D BEST-HNCA. These assignments were the basis to investigate the interaction sites between the protein folding helper enzyme SlyD(1-165) (SlyD*) from Escherichia coli (E. coli) and this kinetic intermediate at a residue resolution. Thus, we investigated the Michaelis-Menten complex of this enzyme reaction, because the NMR data acquisition was performed during the actual catalysis. The interaction surface of the transient folding intermediate is restricted to a region around the peptidyl-prolyl bond (Y38-P39), whose isomerization is catalyzed by SlyD*. The interaction surface regarding SlyD* extends from specific amino acids of the FKBP domain forming the peptidyl-prolyl cis/trans-isomerase active site to almost the entire IF domain. This illustrates an effective interplay between the two functional domains of SlyD* to facilitate protein folding catalysis.     

Kinetic intermediates in the folding of gaseous protein ions characterized by electron capture dissociation mass spectrometry

Alternative mechanisms propose that protein folding in solution proceeds either through specific obligate intermediates or by a multiplicity of routes in a "folding funnel". These questions are examined in the gas phase by using a new method that provides details of the noncovalent binding of solvent-free protein ions. Capture of an electron by a multiply charged cation causes immediate dissociation (ECD) of a backbone bond, but with negligible excitation of noncovalent bonds; thus ECD of a linear protein ion produces two measurable fragment ions only if these are not held together by noncovalent bonds. Thermal unfolding of 9+ ions of cytochrome c proceeds through the separate unfolding of up to 13 backbone regions (represented by 44 bond cleavages) with melting temperatures of <26 to 140 degrees C. An 0.25 s laser IR pulse induces unfolding of 9+ ions in <4 s in six of these regions, followed by their refolding in 2 min. However, for the 15+ ions a laser IR pulse causes slower unfolding through poorly defined intermediates that leads to far more ECD products (63% increase in bond cleavages) after 1 min, even more than heating to 140 degrees C, with refolding to a more compact conformation in 10 min. Random isomerization appears to produce a dynamic mixture of conformers that folds through a variety of pathways to the most stable conformer(s), consistent with a "folding funnel"; this might also be considered as an extension of the classical view to a system with a far smaller free energy change yielding multiple conformers. As cautions to inferring solution conformational structure from gas-phase data, no structural relationship between these gaseous folding intermediates and those in solution is apparent, consistent with reduced hydrophobic bonding and increased electrostatic repulsion. Further, equilibrium folding of gaseous ions can require minutes, and even momentary unfolding of an intermolecular complex during this time can be irreversible.     

New force replica exchange method and protein folding pathways probed by force-clamp technique

We have developed a new extended replica exchange method to study thermodynamics of a system in the presence of external force. Our idea is based on the exchange between different force replicas to accelerate the equilibrium process. This new approach was applied to obtain the force-temperature phase diagram and other thermodynamical quantities of the three-domain ubiquitin. Using the C(alpha)-Go model and the Langevin dynamics, we have shown that the refolding pathways of single ubiquitin depend on which terminus is fixed. If the N end is fixed then the folding pathways are different compared to the case when both termini are free, but fixing the C terminal does not change them. Surprisingly, we have found that the anchoring terminal does not affect the pathways of individual secondary structures of three-domain ubiquitin, indicating the important role of the multidomain construction. Therefore, force-clamp experiments, in which one end of a protein is kept fixed, can probe the refolding pathways of a single free-end ubiquitin if one uses either the polyubiquitin or a single domain with the C terminus anchored. However, it is shown that anchoring one end does not affect refolding pathways of the titin domain I27, and the force-clamp spectroscopy is always capable to predict folding sequencing of this protein. We have obtained the reasonable estimate for unfolding barrier of ubiquitin, using the microscopic theory for the dependence of unfolding time on the external force. The linkage between residue Lys48 and the C terminal of ubiquitin is found to have the dramatic effect on the location of the transition state along the end-to-end distance reaction coordinate, but the multidomain construction leaves the transition state almost unchanged. We have found that the maximum force in the force-extension profile from constant velocity force pulling simulations depends on temperature nonlinearly. However, for some narrow temperature interval this dependence becomes linear, as have been observed in recent experiments.     

Fast folding of a four-helical bundle protein

The FK506-FKBP12 binding-domain of the kinase FRAP (FRB) forms a classic up-down four-helical bundle. The folding pathway of this protein has been investigated using a combination of equilibrium and kinetic studies. The native state of the protein is stable with respect to the unfolded state by some 7 kcal mol(-1) at pH 6.0, 10 degrees C. A kinetic analysis of unfolding and refolding rate constants as a function of chemical denaturant concentration suggests that an intermediate state may be populated during folding at low concentrations of denaturant. The presence of this intermediate state is confirmed by refolding experiments performed in the presence of the hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS). ANS binds to the partially folded intermediate state populated during the folding of FRB and undergoes a large change in fluorescence that can be detected using stopped-flow techniques. Analysis of the kinetic data suggests that the intermediate state is compact and it may even be a misfolded species that has to partially unfold before it can reach the transition state. Folding and unfolding rate constants in water are approximately 150-200 s(-1) and 0.005-0.06 s(-1), respectively, at neutral pH and 10 degrees C. The folding of FRB is somewhat slower than for other all-helical proteins, probably as a consequence of the formation of a metastable intermediate state. The folding rate constant in the absence of any populated intermediate can be estimated to be 8800 s(-1). Despite the presence of an intermediate state, which effectively slows folding, the protein still folds rapidly with a half-life of 5 ms at 10 degrees C. The dependence of the rate constants on denaturant concentration indicates that the transition state for folding is compact with some 80% of the surface area exposed in the unfolded state buried in the transition state. Data presented for FRB is compared with kinetic data obtained for other all-helical proteins.     

Conformational Dynamics of Minimal Elastin‐Like Polypeptides: The Role of Proline Revealed by Molecular Dynamics and Nuclear Magnetic Resonance

Previous molecular dynamics studies of the elastin‐like peptide (ELP) GVG(VPGVG) predict that this ELP undergoes a conformational transition from an open to a more compact closed state upon an increase in temperature. These structural changes occurring in this minimal elastin model at the so‐called inverse temperature transition (ITT), which takes place when elastin is heated to temperatures of about 20–40 ^oC, are investigated further in this work by means of a combined theoretical and experimental approach. To do this, additional extensive classical molecular dynamics (MD) simulations of the capped octapeptide are carried out, analyzed, and compared to data obtained from homonuclear magnetic resonance (NMR) spectroscopy of the same octapeptide. Moreover, in the previous simulations, the proline residue in the ELP is found to act as a hinge, thereby allowing for the large‐amplitude opening and closing conformational motion of the ITT. To explore the role of proline in such elastin repeating units, a point mutant (P5I), which replaces the proline residue with an isoleucine residue, is also investigated using the aforementioned theoretical and experimental techniques. The results show that the site‐directed mutation completely alters the properties of this ELP, thus confirming the importance of the highly conserved proline residue in the ITT. Furthermore, a correlation between the two different methods employed is seen. Both methods predict the mutant ELP to be present in an unstructured form and the wild type ELP to have a β‐turn‐like structure. Finally, the role of the peptidyl cis to trans isomerization of the proline hinge is assessed in detail.     

Involvement of Long‐Lived Intermediate States in the Complex Folding Pathway of the Human Telomeric G‐Quadruplex

The energy landscapes of human telomeric G‐quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real‐time NMR spectroscopy, we investigated the K⁺‐induced folding of the human telomeric DNA sequence 5′‐TTGGG(TTAGGG)₃A‐3′. Three long‐lived states were detected during folding: a major conformation (hybrid‐1), a previously structurally uncharacterized minor conformation (hybrid‐2), and a partially unfolded state. The minor hybrid‐2 conformation is formed faster than the more stable hybrid‐1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin‐like structures.     

The short-lived signaling state of the photoactive yellow protein photoreceptor revealed by combined structural probes

The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 ? upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations.     

The mechanism of GroEL/GroES folding/refolding of protein substrates revisited

The thermodynamics and kinetics of zinc-cytochrome c (ZnCyt c) interactions with Escherichia coli molecular chaperone GroEL (Chaperonin 60; Cpn60) are described. Zinc(II)-porphyrin represents a flexible fluorescent probe for thermodynamic complex formation between GroEL and ZnCyt c, as well as for stopped-flow fluorescence kinetic experiments. Data suggests that GroEL and GroEL/GroES-assisted refolding of unfolded ZnCyt c takes place by a mechanism that is quite close to the Anfinsen Cage hypothesis for molecular chaperone activity. However, even in the presence of ATP, GroEL/GroES-assisted refolding of ZnCyt c takes place at approximately half the rate of refolding of ZnCyt c alone. On the other hand, there is little evidence for refolding behaviour consistent with the Iterative Annealing hypothesis. This includes a complete lack of GroEL or GroEL/GroES-assisted enhancement of refolding rate constant k(2) associated with the unfolding of a putative misfolded state I (Zn) on the pathway to the native state. Reviewing our data in the light of data from other laboratories, we observe that all forward rate enhancements or reductions could be accounted for in terms of thermodynamic coupling (adjusting positions of refolding equilibria) due to binding interactions between GroEL and unfolded protein substrates, driven by thermodynamic considerations. Therefore, we propose that passive kinetic partitioning should be considered the core mechanism of the GroEL/GroES molecular chaperone machinery, wherein the core function is to bind unfolded protein substrates leading to a blockade of aggregation pathways and to increases in molecular flux through productive folding pathway(s).     

Topology-based models and NMR structures in protein folding simulations

Topology-based interaction potentials are simplified models that use the native contacts in the folded structure of a protein to define an energetically unfrustrated folding funnel. They have been widely used to analyze the folding transition and pathways of different proteins through computer simulations. Obviously, they need a reliable, experimentally determined folded structure to define the model interactions. In structures elucidated through NMR spectroscopy, a complex treatment of the raw experimental data usually provides a series of models, a set of different conformations compatible with the available experimental data. Here, we use an efficient coarse-grained simulation technique to independently consider the contact maps from every different NMR model in a protein whose structure has been resolved by the use of NMR spectroscopy. For lambda-Cro repressor, a homodimeric protein, we have analyzed its folding characteristics with a topology-based model. We have focused on the competition between the folding of the individual chains and their binding to form the final quaternary structure. From 20 different NMR models, we find a predominant three-state folding behavior, in agreement with experimental data on the folding pathway for this protein. Individual NMR models, however, show distinct characteristics, which are analyzed both at the level of the interplay between tertiary/quaternary structure formation and also regarding the thermal stability of the tertiary structure of every individual chain.     

The protein folding network indicates that the ultrafast folding mutant of villin headpiece subdomain has a deeper folding funnel

Protein folding is a dynamic process with continuous transitions among different conformations. In this work, the dynamics in the protein folding network of villin headpiece subdomain (HP35) has been investigated based on multiple reversible folding trajectories of HP35 and its ultrafast folding mutant where sub-angstrom folding was achieved. The four folding states were clearly separated on the network, validating the classification of the states. Examination of the eight conformers with different formation of the individual helices revealed high plasticity of the three helices in all the four states. A consistent feature between the wild type and mutant protein is the dominant conformer 111 (all three helices formed) in the folded state and conformers 111 and 011 (helices II and III formed) in the major intermediate state, indicating the critical role of helices II and III in the folding mechanism. When compared to the wild type, the folding landscape of the ultrafast folding mutant exhibited a deeper folding funnel towards the folded state. The very beginning of the folding (0-10 ns) was very similar for both protein variants but it soon diverged and displayed different folding pathways. Although going through the major intermediate state is the dominant pathway for both, it was also observed that some folding went through the minor intermediate state for the mutant. The intriguing difference resulting from the mutation at two residues in helix III has been carefully analyzed and discussed in details.     

Modulation of structure and dynamics by disulfide bond formation in unfolded states

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.     

Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding

Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.     

Mechanistic imperatives for aldose-ketose isomerization in water: specific, general base- and metal ion-catalyzed isomerization of glyceraldehyde with proton and hydride transfer.

The deuterium enrichment of dihydroxyacetone obtained from the aldose-ketose isomerization of D,L-glyceraldehyde in D(2)O at 25 degrees C was determined by (1)H NMR spectroscopy from the integrated areas of the signals for the alpha-CH(2) and alpha-CHD groups of the product. One mole equivalent of deuterium is incorporated into the product when the isomerization is carried out in 150 mM pyrophosphate buffer at pD 8.4, but only 0.6 mol equiv of deuterium is incorporated into the product of isomerization in the presence of 0.01 M deuterioxide ion, so that 40% of the latter isomerization reaction proceeds by the intramolecular transfer of hydride ion. Several pathways were identified for catalysis of the isomerization of glyceraldehyde to give dihydroxyacetone. The isomerization with hydride transfer is strongly catalyzed by added Zn(2+). Deprotonation of glyceraldehyde is rate-determining for isomerization with proton transfer, and this proton-transfer reaction is catalyzed by Br?nsted bases. Proton transfer also occurs by a termolecular pathway with catalysis by the combined action of Br?nsted bases and Zn(2+). These results show that there is no large advantage to the spontaneous isomerization of glyceraldehyde in alkaline solution with either proton or hydride transfer, and that effective catalytic pathways exist to stabilize the transition states for both of these reactions in water. The existence of separate enzymes that catalyze the isomerization of sugars with hydride transfer and the isomerization of sugar phosphates with proton transfer is proposed to be a consequence of the lack of any large advantage to reaction by either of these pathways for the corresponding nonenzymatic isomerization in water.     

Protein folding kinetics under force from molecular simulation

Despite a large number of studies on the mechanical unfolding of proteins, there are still relatively few successful attempts to refold proteins in the presence of a stretching force. We explore refolding kinetics under force using simulations of a coarse-grained model of ubiquitin. The effects of force on the folding kinetics can be fitted by a one-dimensional Kramers theory of diffusive barrier crossing, resulting in physically meaningful parameters for the height and location of the folding activation barrier. By comparing parameters obtained from pulling in different directions, we find that the unfolded state plays a dominant role in the refolding kinetics. Our findings explain why refolding becomes very slow at even moderate pulling forces and suggest how it could be practically observed in experiments at higher forces.     

Reversible Unfolding–Refolding of Rubredoxin: A Single‐Molecule Force Spectroscopy Study

In metalloproteins, metal centers serve as active sites for a range of functional purposes and as important structural elements to facilitate protein folding and assembly. It is challenging to observe the reversible unfolding and refolding of metalloproteins because of a loss or decomposition of the metal center. Here, the reversible unfolding–refolding of the iron–sulfur protein rubredoxin was observed directly using single‐molecule force spectroscopy. The results demonstrate that the iron can remain attached to the CXXC motif when rubredoxin is unfolded. Upon relaxation, the unfolded rubredoxin can refold into its native holo state with the reconstituted FeS₄ center. The possible loss of iron from the unfolded protein prevents rubredoxin from refolding into its native holo state. These results demonstrated that unfolding of rubredoxin is reversible, as long as the iron remains attached, and provide experimental evidence for the iron‐priming mechanism for the folding of rubredoxin.     

Rerouting the folding pathway of the Notch ankyrin domain by reshaping the energy landscape

The modular nature of repeat proteins has made them a successful target for protein design. Ankyrin repeat, TPR, and leucine rich repeat domains that have been designed solely on consensus information have been shown to have higher thermostability than their biological counterparts. We have previously shown that we can reshape the energy landscape of a repeat protein by adding multiple C-terminal consensus ankyrin repeats to the five N-terminal repeats of the Notch ankyrin domain. Here we explore how the folding mechanism responds to reshaping of the energy landscape. We have used analogous substitutions of a conserved alanine with glycine in each repeat to determine the distribution of structure in the transition state ensembles of constructs containing one (Nank1-5C1) and two consensus (Nank1-5C2) ankyrin repeats. Whereas folding of the wild-type Notch ankyrin domain is slowed by substitutions in its central repeats, (1) folding of Nank1-5C1 and Nank1-5C2 is slowed by substitutions in the C-terminal repeats. Thus, the addition of C-terminal stabilizing repeats shifts the transition state ensemble toward the C-terminal repeats, rerouting the folding pathway of the ankyrin repeat domain. These findings indicate that, for the Notch ankyrin domain, folding pathways are selected based on local energetics.     

Tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase for simultaneous reversed‐phase/hydrophilic interaction mixed‐mode chromatography

A new water‐soluble tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase was prepared straightforwardly by an indirect method and characterized by elemental analysis, energy dispersive Spectrometry, solid‐state ¹³C NMR spectroscopy, Fourier‐transform infrared spectroscopy, and thermogravimetric analysis. Due to the simultaneous introduction of polar tetra‐proline and nonpolar calix[4]arene, the developed column possessing a double retention mode of reverse‐phase liquid chromatography and hydrophilic interaction liquid chromatography. A series of hydrophobic and hydrophilic test samples, including nucleosides and nucleotides, amines, monosubstituted benzenes, chiral compounds, and phenols, were used to evaluate the developed stationary phase. A rapid separation capability, high separation efficiency, and selectivity were achieved based on the multiple interactions between solutes and tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase. Moreover, the developed stationary phase was further used to detect and separate hexamethylenetetramine in rice flour. All the results indicated the potential merits of the developed stationary phase for simultaneous separation of complex hydrophobic and hydrophilic samples with high selectivity.     

The effect of a trans-locked Gly-Pro alkene isostere on collagen triple helix stability

An alkene isostere of Gly-trans-Pro was synthesized and incorporated into a host Ac-(Gly-Pro-Hyp)8-Gly-Gly-Tyr-NH2 peptide to investigate the effect of locking a proline amide bond. Proline amide bond isomerization is the slow step in collagen folding. By locking the amide, we hypothesized an increase in stability of the collagen triple helix. The substitution instead destabilized the collagen host peptide. The Tm value of the host control peptide was 50.0 degrees C, while the peptide containing the isostere, Ac-(Gly-Pro-Hyp)3-Gly-psi[(E)CH C]-Pro-Hyp-(Gly-Pro-Hyp)4-Gly-Gly-Tyr-NH2, had a Tm value of 28.3 degrees C. There are clearly factors that contribute to collagen stability and folding that we do not yet understand.     

Retinal Conformation and Dynamics in Activation of Rhodopsin Illuminated by Solid‐state 2H NMR Spectroscopy†

Solid‐state NMR spectroscopy gives a powerful avenue for investigating G protein‐coupled receptors and other integral membrane proteins in a native‐like environment. This article reviews the use of solid‐state ²H NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site‐specific ²H NMR labels have been introduced into three regions (methyl groups) of retinal that are crucially important for the photochemical function of rhodopsin. Despite its phenomenal stability ²H NMR spectroscopy indicates retinal undergoes rapid fluctuations within the protein binding cavity. The spectral lineshapes reveal the methyl groups spin rapidly about their three‐fold (C₃) axes with an order parameter for the off‐axial motion of For the dark state, the ²H NMR structure of 11‐cis‐retinal manifests torsional twisting of both the polyene chain and the β‐ionone ring due to steric interactions of the ligand and the protein. Retinal is accommodated within the rhodopsin binding pocket with a negative pretwist about the C11=C12 double bond. Conformational distortion explains its rapid photochemistry and reveals the trajectory of the 11‐cis to trans isomerization. In addition, ²H NMR has been applied to study the retinylidene dynamics in the dark and light‐activated states. Upon isomerization there are drastic changes in the mobility of all three methyl groups. The relaxation data support an activation mechanism whereby the β‐ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the β‐ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid‐state ²H NMR thus provides information about the flow of energy that triggers changes in hydrogen‐bonding networks and helix movements in the activation mechanism of the photoreceptor.