Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.

Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

A fit-less approach to the elasticity of the handles in optical tweezers experiments.

The elastic properties of the double-stranded DNA handles used in optical tweezers experiments on biomolecules are customarily modeled by an extensible worm-like chain model. Fitting such a model to experimental data, however, is no trivial task, as the function depends on four parameters in a highly non-linear fashion. We hereby propose a method to bypass the fitting procedure and obtain an empirical force vs. extension curve that accurately reproduces the elasticity of the handles.

Effects of Ligand Binding on the Energy Landscape of Acyl-CoA-Binding Protein.

Binding of ligands is often crucial for function yet the effects of ligand binding on the mechanical stability and energy landscape of proteins are incompletely understood. Here, we use a combination of single-molecule optical tweezers and MD simulations to investigate the effect of ligand binding on the energy landscape of acyl-coenzyme A (CoA)-binding protein (ACBP). ACBP is a topologically simple and highly conserved four-α-helix bundle protein that acts as an intracellular transporter and buffer for fatty-acyl-CoA and is active in membrane assembly. We have previously described the behavior of ACBP under tension, revealing a highly extended transition state (TS) located almost halfway between the unfolded and native states. Here, we performed force-ramp and force-jump experiments, in combination with advanced statistical analysis, to show that octanoyl-CoA binding increases the activation free energy for the unfolding reaction of ACBP without affecting the position of the transition state along the reaction coordinate. It follows that ligand binding enhances the mechanical resistance and thermodynamic stability of the protein, without changing its mechanical compliance. Steered molecular dynamics simulations allowed us to rationalize the results in terms of key interactions that octanoyl-CoA establishes with the four α-helices of ACBP and showed that the unfolding pathway is marginally affected by the ligand. The results show that ligand-induced mechanical stabilization effects can be complex and may prove useful for the rational design of stabilizing ligands.

Bio-Molecular Applications of Recent Developments in Optical Tweezers.

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT's resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

Single-molecule folding mechanisms of the apo- and Mg(2+)-bound states of human neuronal calcium sensor-1.

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca(2+) concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca(2+)-activated state of NCS proteins, little is known about the conformational dynamics of the Mg(2+)-bound and apo states, both of which are populated, at least transiently, at resting Ca(2+) conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg(2+) and in the absence of divalent ions. Under tension, the Mg(2+)-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca(2+)-bound state, the presence of Mg(2+) allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.

Dynamics of a homogeneous active dumbbell system.

We analyze the dynamics of a two-dimensional system of interacting active dumbbells. We characterize the mean-square displacement, linear response function, and deviation from the equilibrium fluctuation-dissipation theorem as a function of activity strength, packing fraction, and temperature for parameters such that the system is in its homogeneous phase. While the diffusion constant in the last diffusive regime naturally increases with activity and decreases with packing fraction, we exhibit an intriguing nonmonotonic dependence on the activity of the ratio between the finite-density and the single-particle diffusion constants. At fixed packing fraction, the time-integrated linear response function depends nonmonotonically on activity strength. The effective temperature extracted from the ratio between the integrated linear response and the mean-square displacement in the last diffusive regime is always higher than the ambient temperature, increases with increasing activity, and, for small active force, monotonically increases with density while for sufficiently high activity it first increases and next decreases with the packing fraction. We ascribe this peculiar effect to the existence of finite-size clusters for sufficiently high activity and density at the fixed (low) temperatures at which we worked. The crossover occurs at lower activity or density the lower the external temperature. The finite-density effective temperature is higher (lower) than the single dumbbell one below (above) a crossover value of the Péclet number.

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1.

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.

Single-molecule folding mechanism of an EF-hand neuronal calcium sensor.

EF-hand calcium sensors respond structurally to changes in intracellular Ca(2+) concentration, triggering diverse cellular responses and resulting in broad interactomes. Despite impressive advances in decoding their structure-function relationships, the folding mechanism of neuronal calcium sensors is still elusive. We used single-molecule optical tweezers to study the folding mechanism of the human neuronal calcium sensor 1 (NCS1). Two intermediate structures induced by Ca(2+) binding to the EF-hands were observed during refolding. The complete folding of the C domain is obligatory for the folding of the N domain, showing striking interdomain dependence. Molecular dynamics results reveal the atomistic details of the unfolding process and rationalize the different domain stabilities during mechanical unfolding. Through constant-force experiments and hidden Markov model analysis, the free energy landscape of the protein was reconstructed. Our results emphasize that NCS1 has evolved a remarkable complex interdomain cooperativity and a fundamentally different folding mechanism compared to structurally related proteins.

Measurement of work in single-molecule pulling experiments.

A main goal of single-molecule experiments is to evaluate equilibrium free energy differences by applying fluctuation relations to repeated work measurements along irreversible processes. We quantify the error that is made in a free energy estimate by means of the Jarzynski equality when the accumulated work expended on the whole system (including the instrument) is erroneously replaced by the work transferred to the subsystem consisting of the sole molecular construct. We find that the error may be as large as 100%, depending on the number of experiments and on the bandwidth of the data acquisition apparatus. Our theoretical estimate is validated by numerical simulations and pulling experiments on DNA hairpins using optical tweezers.

Recovery of free energy branches in single molecule experiments.

We present a method for determining the free energy of coexisting states from irreversible work measurements. Our approach is based on a fluctuation relation that is valid for dissipative transformations in partially equilibrated systems. To illustrate the validity and usefulness of the approach, we use optical tweezers to determine the free energy branches of the native and unfolded states of a two-state molecule as a function of the pulling control parameter. We determine, within 0.6kBT accuracy, the transition point where the free energies of the native and the unfolded states are equal.

Supersymmetric Langevin equation to explore free-energy landscapes.

The recently discovered supersymmetric generalizations of the Langevin dynamics and Kramers equation can be utilized for the exploration of free-energy landscapes of systems whose large time-scale separation hampers the usefulness of standard molecular dynamics techniques. The first realistic application is here presented. The system chosen is a minimalist model for a short alanine peptide exhibiting a helix-coil transition.

Hamiltonian dynamics of homopolymer chain models.

The Hamiltonian dynamics of chains of nonlinearly coupled particles is numerically investigated in two and three dimensions. Simple, off-lattice homopolymer models are used to represent the interparticle potentials. Time averages of observables numerically computed along dynamical trajectories are found to reproduce results given by the statistical mechanics of homopolymer models. The dynamical treatment, however, indicates a nontrivial transition between regimes of slow and fast phase space mixing. Such a transition is inaccessible to a statistical mechanical treatment and reflects a bimodality in the relaxation of time averages to corresponding ensemble averages. It is also found that a change in the energy dependence of the largest Lyapunov exponent indicates the Theta transition between filamentary and globular polymer configurations, clearly detecting the transition even for a finite number of particles.

Topological origin of the phase transition in a model of DNA denaturation.

The hypothesis that phase transitions originate from some topological change of the critical level hypersurface of the potential energy receives direct evident support by our study of the Bishop-Peyrard model of DNA thermal denaturation.