Changbong Hyeon

Capturing the essence of folding and functions of biomolecules using coarse-grained models

The distances over which biological molecules and their complexes can function range from a few nanometres, in the case of folded structures, to millimetres, for example, during chromosome organization. Describing phenomena that cover such diverse length, and also time, scales requires models that capture the underlying physics for the particular length scale of interest. Theoretical ideas, in particular, concepts from polymer physics, have guided the development of coarse-grained models to study folding of DNA, RNA and proteins. More recently, such models and their variants have been applied to the functions of biological nanomachines. Simulations using coarse-grained models are now poised to address a wide range of problems in biology.

Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations

Nanomanipulation of biomolecules by using single-molecule methods and computer simulations has made it possible to visualize the energy landscape of biomolecules and the structures that are sampled during the folding process. We use simulations and single-molecule force spectroscopy to map the complex energy landscape of GFP that is used as a marker in cell biology and biotechnology. By engineering internal disulfide bonds at selected positions in the GFP structure, mechanical unfolding routes are precisely controlled, thus allowing us to infer features of the energy landscape of the wild-type GFP. To elucidate the structures of the unfolding pathways and reveal the multiple unfolding routes, the experimental results are complemented with simulations of a self-organized polymer (SOP) model of GFP. The SOP representation of proteins, which is a coarse-grained description of biomolecules, allows us to perform forced-induced simulations at loading rates and time scales that closely match those used in atomic force microscopy experiments. By using the combined approach, we show that forced unfolding of GFP involves a bifurcation in the pathways to the stretched state. After detachment of an N-terminal α-helix, unfolding proceeds along two distinct pathways. In the dominant pathway, unfolding starts from the detachment of the primary N-terminal β-strand, while in the minor pathway rupture of the last, C-terminal β-strand initiates the unfolding process. The combined approach has allowed us to map the features of the complex energy landscape of GFP including a characterization of the structures, albeit at a coarse-grained level, of the three metastable intermediates.

Theoretical Perspectives on Protein Folding

Understanding how monomeric proteins fold under in vitro conditions is crucial to describing their functions in the cellular context. Significant advances in theory and experiments have resulted in a conceptual framework for describing the folding mechanisms of globular proteins. The sizes of proteins in the denatured and folded states, cooperativity of the folding transition, dispersions in the melting temperatures at the residue level, and timescales of folding are, to a large extent, determined by N, the number of residues. The intricate details of folding as a function of denaturant concentration can be predicted by using a novel coarse-grained molecular transfer model. By watching one molecule fold at a time, using single-molecule methods, investigators have established the validity of the theoretically anticipated heterogeneity in the folding routes and the N-dependent timescales for the three stages in the approach to the native state. Despite the successes of theory, of which only a few examples are documented here, we conclude that much remains to be done to solve the protein folding problem in the broadest sense.

Efficient functional delivery of siRNA using mesoporous silica nanoparticles with ultralarge pores.

Among various nanoparticles, mesoporous silica nanoparticles (MSNs) have attracted extensive attention for developing efficient drug-delivery systems, mostly due to their high porosity and biocompatibility. However, due to the small pore size, generally below 5 nm in diameter, potential drugs that are loaded into the pore have been limited to small molecules. Herein, a small interfering RNA (siRNA) delivery strategy based on MSNs possessing pores with an average diameter of 23 nm is presented. The siRNA is regarded as a powerful gene therapeutic agent for treatment of a wide range of diseases by enabling post-transcriptional gene silencing, so-called RNA interference. Highly efficient, sequence-specific, and technically very simple target gene knockdown is demonstrated using MSNs with ultralarge pores of size 23 nm in vitro and in vivo without notable cytotoxicity.

Dynamics of allosteric transitions in GroEL

The chaperonin GroEL-GroES, a machine that helps proteins to fold, cycles through a number of allosteric states, the T state, with high affinity for substrate proteins, the ATP-bound R state, and the R″ (GroEL–ADP–GroES) complex. Here, we use a self-organized polymer model for the GroEL allosteric states and a general structure-based technique to simulate the dynamics of allosteric transitions in two subunits of GroEL and the heptamer. The T → R transition, in which the apical domains undergo counterclockwise motion, is mediated by a multiple salt-bridge switch mechanism, in which a series of salt-bridges break and form. The initial event in the R → R″ transition, during which GroEL rotates clockwise, involves a spectacular outside-in movement of helices K and L that results in K80-D359 salt-bridge formation. In both the transitions there is considerable heterogeneity in the transition pathways. The transition state ensembles (TSEs) connecting the T, R, and R″ states are broad with the TSE for the T → R transition being more plastic than the R → R″ TSE.

Can energy landscape roughness of proteins and RNA be measured by using mechanical unfolding experiments

By considering temperature effects on the mechanical unfolding rates of proteins and RNA, whose energy landscape is rugged, the question posed in the title is answered in the affirmative. Adopting a theory by Zwanzig [Zwanzig, R. (1988) Proc. Natl. Acad. Sci. USA 85, 2029-2030], we show that, because of roughness characterized by an energy scale ε, the unfolding rate at constant force is retarded. Similarly, in nonequilibrium experiments done at constant loading rates, the most probable unfolding force increases because of energy landscape roughness. The effects are dramatic at low temperatures. Our analysis suggests that, by using temperature as a variable in mechanical unfolding experiments of proteins and RNA, the ruggedness energy scale ε, can be directly measured.

Dynamic Ca2+-Dependent Stimulation of Vesicle Fusion by Membrane-Anchored Synaptotagmin 1

A Trick of the Tail The synaptic vesicle protein, synaptotagmin 1 (Syt1), acts as the main Ca2+-dependent switch for neurotransmitter release. In vitro studies of the truncated Syt1, which lacks the transmembrane domain, have unveiled the fusion-triggering mechanism of Syt1. However, in vitro approaches using the full-length, membrane-anchored Syt1 have not only failed to recapitulate Ca2+-triggered membrane fusion, but could even inhibit vesicle fusion. In contrast, the membrane anchor is conserved across the Syt family, suggesting a critical functional role for the membrane anchor. Now, using a single vesicle fusion assay, H.-K. Lee et al. (p. 760) show that the membrane anchor is indeed essential for Syt1 to induce physiological rates of Ca2+-induced vesicle fusion on a 100-millisecond time scale. A synaptic vesicle protein must be membrane-anchored to stimulate fusion in vitro at physiological Ca2+ concentrations. In neurons, synaptotagmin 1 (Syt1) is thought to mediate the fusion of synaptic vesicles with the plasma membrane when presynaptic Ca2+ levels rise. However, in vitro reconstitution experiments have failed to recapitulate key characteristics of Ca2+-triggered membrane fusion. Using an in vitro single-vesicle fusion assay, we found that membrane-anchored Syt1 enhanced Ca2+ sensitivity and fusion speed. This stimulatory activity of membrane-anchored Syt1 dropped as the Ca2+ level rose beyond physiological levels. Thus, Syt1 requires the membrane anchor to stimulate vesicle fusion at physiological Ca2+ levels and may function as a dynamic presynaptic Ca2+ sensor to control the probability of neurotransmitter release.

Internal strain regulates the nucleotide binding site of the kinesin leading head

In the presence of ATP, kinesin proceeds along the protofilament of microtubule by alternated binding of two motor domains on the tubulin binding sites. Because the processivity of kinesin is much higher than other motor proteins, it has been speculated that there exists a mechanism for allosteric regulation between the two monomers. Recent experiments suggest that ATP binding to the leading head (L) domain in kinesin is regulated by the rearward strain built on the neck-linker. We test this hypothesis by explicitly modeling a Cα-based kinesin structure whose motor domains are bound on the tubulin binding sites. The equilibrium structures of kinesin on the microtubule show disordered and ordered neck-linker configurations for the L and trailing head, respectively. The comparison of the structures between the two heads shows that several native contacts present at the nucleotide binding site in the L are less intact than those in the binding site of the rear head. The network of native contacts obtained from this comparison provides the internal tension propagation pathway, which leads to the disruption of the nucleotide binding site in the L. Also, using an argument based on polymer theory, we estimate the internal tension built on the neck-linker to be f ≈ 12–15 pN. Both of these conclusions support the experimental hypothesis.

Mechanical control of the directional stepping dynamics of the kinesin motor

Among the multiple steps constituting the kinesin mechanochemical cycle, one of the most interesting events is observed when kinesins move an 8-nm step from one microtubule (MT)-binding site to another. The stepping motion that occurs within a relatively short time scale (≈100 μs) is, however, beyond the resolution of current experiments. Therefore, a basic understanding to the real-time dynamics within the 8-nm step is still lacking. For instance, the rate of power stroke (or conformational change) that leads to the undocked-to-docked transition of neck-linker is not known, and the existence of a substep during the 8-nm step still remains a controversial issue in the kinesin community. By using explicit structures of the kinesin dimer and the MT consisting of 13 protofilaments, we study the stepping dynamics with varying rates of power stroke (kp). We estimate that kp−1 ≲ 20 μs to avoid a substep in an averaged time trace. For a slow power stroke with kp−1 > 20 μs, the averaged time trace shows a substep that implies the existence of a transient intermediate, which is reminiscent of a recent single-molecule experiment at high resolution. We identify the intermediate as a conformation in which the tethered head is trapped in the sideway binding site of the neighboring protofilament. We also find a partial unfolding (cracking) of the binding motifs occurring at the transition state ensemble along the pathways before binding between the kinesin and MT.

Measuring the energy landscape roughness and the transition state location of biomolecules using single molecule mechanical unfolding experiments

Single molecule mechanical unfolding experiments are beginning to provide profiles of the complex energy landscape of biomolecules. In order to obtain reliable estimates of the energy landscape characteristics it is necessary to combine the experimental measurements (the force?extension curves, the mechanical unfolding trajectories, force or loading rate dependent unfolding rates) with sound theoretical models and simulations. Here, we show how by using temperature as a variable in mechanical unfolding of biomolecules in laser optical tweezer or AFM experiments the roughness of the energy landscape can be measured without making any assumptions about the underlying reaction coordinate. The efficacy of the formalism is illustrated by reviewing experimental results that have directly measured roughness in a protein?protein complex. The roughness model can also be used to interpret experiments on forced unfolding of proteins in which temperature is varied. Estimates of other aspects of the energy landscape such as free energy barriers or the transition state (TS) locations could depend on the precise model used to analyse the experimental data. We illustrate the inherent difficulties in obtaining the transition state location from loading rate or force dependent unfolding rates. Because the transition state moves as the force or the loading rate is varied it is in general difficult to invert the experimental data unless the curvature at the top of the one dimensional free energy profile is large, i.e.?the barrier is sharp. The independence of the TS location of the force holds good only for brittle or hard biomolecules whereas the TS location changes considerably if the molecule is soft or plastic. We also comment on the usefulness of extension of the molecule as a surrogate reaction coordinate especially in the context of force-quench refolding of proteins and RNA.