In an editorial for a Special Issue, Nussinov and Wolynes explored the energy landscapes of biomolecular function, questioning whether they constituted a second molecular biology revolution. With more than a decade having passed and science having progressed significantly, we revisit this question. Statistical energy landscapes not only visualize folding funnels but also quantify the likelihoods of different states, embodying the foundational physical-chemical principles of protein actions. Building upon the theory of energy landscapes, the conformational selection and population shift paradigm posited that since all functional conformations already pre-exist in a dynamic equilibrium, a ligand ‘selects’ and stabilizes a state from this pre-existing pool, resulting in re-equilibration, or shift, of the population. The principle that it established — that function harnesses transitions between pre-existing conformations — revolutionized the understanding of allostery and, broadly, regulation. This paradigm challenged and superseded the decades-old, albeit persisting, belief of only one (or two; ‘open’ and ‘closed’) protein conformations. It also indicates that for engineered proteins to exert effective function, we must account for the timescales of flipping between energy landscape states, for example, by tuning the barrier heights. Returning to the question of whether landscapes constituted a second biomolecular biology revolution, we consider their bedrock contributions, which are far beyond the original protein folding funnels. They established the principle of multiple dynamic conformational states ‘jumping’ over barriers during population shifts. By leveraging core concepts like conformational ensembles, modern molecular biology has achieved breakthroughs such as next-generation allosteric drugs, indeed leading to a transformative era in molecular science.

The TRiC/CCT complex assists in the folding of approximately 10% of cytosolic proteins through an ATP-driven conformational cycle, playing a crucial role in maintaining protein homeostasis. Despite our understanding of ATP-driven TRiC ring closing and substrate folding, the process and mechanisms underlying TRiC ring-opening and substrate release remain largely unexplored. In this study, by determining an ensemble of cryo-EM structures of yeast TRiC in the presence of ADP, including three intermediate transition states, we present a comprehensive picture of the TRiC ring-opening process. During this process, CCT3 detects the loss of γ-phosphate and initiates with the dynamics of its apical protrusion, and expands to the outward leaning of the consecutive CCT6/8/7/5 subunits. This is followed by significant movements of CCT2, CCT4, and especially CCT1 subunits, resulting in the opening of the TRiC rings. We also observed an unforeseen temporary separation between the two rings in the CCT2 side, coordinating the release of the originally locked CCT4 N-terminus, which potentially participates in the ring-opening process. Collectively, our study reveals a stepwise TRiC ring-opening mechanism, provides a comprehensive view of the TRiC conformational landscape, and sheds lights on its subunit specificity in sensing nucleotide status and substrate release. Our findings deepen our understanding of protein folding assisted by TRiC and may inspire new strategies for the diagnosis and treatment of related diseases.

Conformational changes are often required for the biological function of RNA molecules. In the Tetrahymena group I ribozyme reaction, a conformational change has been suggested to occur upon binding of the oligonucleotide substrate (S) or the guanosine nucleophile (G), leading to stronger binding of the second substrate. Recent work showed that the two substrates are bridged by a metal ion that coordinates both the nonbridging reactive phosphoryl oxygen of S and the 2′-OH of G. These results suggest that the energy from the metal ion[bull]substrate interactions is used to drive the proposed conformational change. In this work, we provide an experimental test for this model. The results provide strong support for the proposed conformational change and for a central role of the bridging metal ion in this change. The results from this work, combined with previous data, allow construction of a two-state model that quantitatively accounts for all of the observations in this and previous work. This model provides a conceptual and quantitative framework that will facilitate understanding and further probing of the energetic and structural features of this conformational change and its role in catalysis.

The hairpin ribozyme is a short endonucleolytic RNA motif isolated from a family of related plant virus satellite RNAs. It consists of two independently folding domains, each comprising two Watson–Crick helices flanking a conserved internal loop. The domains need to physically interact (dock) for catalysis of site-specific cleavage and ligation reactions. Using tapping-mode atomic force microscopy in aqueous buffer solution, we were able to produce high quality images of individual hairpin ribozyme molecules with extended terminal helices. Three RNA constructs with either the essential cleavage site guanosine or a detrimental adenosine substitution and with or without a 6-nt insertion to confer flexibility to the interdomain hinge show structural differences that correlate with their ability to form the active docked conformation. The observed contour lengths and shapes are consistent with previous bulk-solution measurements of the transient electric dichroism decays for the same RNA constructs. The active docked construct appears as an asymmetrically docked conformation that might be an indication of a more complicated docking event than a simple collapse around the interdomain hinge.

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N-terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N-terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.

The role of the switch II region in the conformational transition of activation of Ha-ras-p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Diaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434–451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3− to the GDP-p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction.

The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X-ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800–816). This form of TrxR, denoted FO, permits the reaction of enzyme-bound reduced FAD with a redox-active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X-ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34° “butterfly” bend about the N(5)–N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15–28° for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side-chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.

This work experimentally confirms the pathway of activation of Ha-ras-p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434–451). The process can be studied experimentally by analyzing the binding of BeF3− to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138–23143). Two mutants, V29G and I36G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.

The serine protease subtilisin BPN′ is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the kcat for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the kcat for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 Å resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180° around the Cβ-Cγ bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents.

NMR spectroscopic analysis of the C-terminal Kunitz domain fragment (α3(VI)) from the human α3-chain of type VI collagen has revealed that the side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X-ray crystallographic structure, while the minor conformation (m) cannot structurally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conformations of the Trp21 side chain obtained by energy minimization identified two energy minima: One corresponding to the conformation of Trp21 observed in the X-ray crystallographic structure and solution structure of α3(VI) (the M conformation) and the second corresponding to the m conformation predicted by NMR spectroscopy. A transition pathway between the M and m conformation is suggested. The free-energy difference between the two conformers obtained by the thermodynamic integration method is calculated to 1.77 ± 0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformers of the α3(VI) molecule were investigated by molecular dynamic. Essential dynamics analysis of the two resulting 800 ps trajectories reveals that when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are observed in the mobility of the binding loop (residues Thr13–Ile18), which is more flexible in the m conformation than in the M conformation. This suggests that the reorientation of Trp21 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding loop and Trp21.

The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and −33°C. Eyring plots are linear down to −27°C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.