Enzyme-DNA chimeras: Construction, allostery, applications.

We describe the operational principle, synthesis, and applications of the enzyme-DNA chimeras. These are supramolecular constructions where a DNA spring is coupled to an enzyme and introduces artificial allosteric control of the enzyme. This method is universal and can be applied to various enzymes and proteins. In addition, this method is versatile as the stresses applied by the DNA spring on the enzymes can be fine-tuned semi-continuously and thus their enzymatic activities can be modulated gradually. We give detailed protocols for the synthesis of these molecules. Summarizing our experience with different enzymes, we explain their use for fundamental studies of conformational plasticity, as well as the potential as molecular probes.

Analog control with two Artificial Axons.

The artificial axon is a recently introduced synthetic assembly of supported lipid bilayers and voltage gated ion channels, displaying the basic electrophysiology of nerve cells. Here we demonstrate the use of two artificial axons as control elements to achieve a simple task. Namely, we steer a remote control car towards a light source, using the sensory input dependent firing rate of the axons as the control signal for turning left or right. We present the result in the form of the analysis of a movie of the car approaching the light source. In general terms, with this work we pursue a constructivist approach to exploring the nexus between machine language at the nerve cell level and behavior.

Dissipation at the angstrom scale: Probing the surface and interior of an enzyme.

Pursuing a materials science approach to understanding the deformability of enzymes, we introduce measurements of the phase of the mechanical response function within the nanorheology paradigm. Driven conformational motion of the enzyme is dissipative as characterized by the phase measurements. The dissipation originates both from the surface hydration layer and the interior of the molecule, probed by examining the effect of point mutations on the mechanics. We also document changes in the mechanics of the enzyme examined, guanylate kinase, upon binding its four substrates. GMP binding stiffens the molecule, ATP and ADP binding softens it, while there is no clear mechanical signature of GDP binding. A hyperactive two-Gly mutant is found to possibly trade specificity for speed. Global deformations of enzymes are shown to be dependent on both hydration layer and polypeptide chain dynamics.

Mechanical properties of BiP protein determined by nano-rheology.

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (KD ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.

Identifying Chaotropic and Kosmotropic Agents by Nanorheology.

Chemical agents are classified into chaotropes (disorder inducing) and kosmotropes (order inducing) based on their ability to destabilize or stabilize, respectively, the structure of hydrated macromolecules and their solutions. Here, we examine the effect of such agents on the mechanical stiffness of the hydration layer of proteins, measured by nanorheology. We examine four different agents and conclude that chaotropic substances induce the overall softening of the protein-hydration layer system, whereas the kosmotropic substances induce stiffening. Specifically, with glucose and trifluoroethanol, two known kosmotropic agents, we observe the stiffening of the hydration layer. In contrast, with guanidine hydrochloride and urea, known kaotropic agents, we observe softening. Thus, the viscoelastic mechanics of the folded, hydrated protein provides an experimental measure of the effect that chaotropes and kosmotropes have on the dynamics.

Toward a Minimal Artificial Axon.

The electrophysiology of action potentials is usually studied in neurons, through relatively demanding experiments which are difficult to scale up to a defined network. Here we pursue instead the minimal artificial system based on the essential biological components-ion channels and lipid bilayers-where action potentials can be generated, propagated, and eventually networked. The fundamental unit is the classic supported bilayer: a planar bilayer patch with embedded ion channels in a fluidic environment where an ionic gradient is imposed across the bilayer. Two such units electrically connected form the basic building block for a network. The system is minimal in that we demonstrate that one kind of ion channel and correspondingly a gradient of only one ionic species is sufficient to generate an excitable system which shows amplification and threshold behavior.

Artificial phosphorylation sites modulate the activity of a voltage-gated potassium channel.

The KvAP potassium channel is representative of a family of voltage-gated ion channels where the membrane potential is sensed by a transmembrane helix containing several positively charged arginines. Previous work by Wang and Zocchi [A. Wang and G. Zocchi, PLoS ONE 6, e18598 (2011)] showed how a negatively charged polyelectrolyte attached in proximity to the voltage sensing element can bias the opening probability of the channel. Here we introduce three phosphorylation sites at the same location and show that the response curve of the channel shifts by about 20 mV upon phosphorylation, while other characteristics such as the single-channel conductance are unaffected. In summary, we construct an artificial phosphorylation site which confers allosteric regulation to the channel.

Dissipative dynamics of enzymes.

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

Asymmetric effect of mechanical stress on the forward and reverse reaction catalyzed by an enzyme.

The concept of modulating enzymatic activity by exerting a mechanical stress on the enzyme has been established in previous work. Mechanical perturbation is also a tool for probing conformational motion accompanying the enzymatic cycle. Here we report measurements of the forward and reverse kinetics of the enzyme Guanylate Kinase from yeast (Saccharomyces cerevisiae). The enzyme is held in a state of stress using the DNA spring method. The observation that mechanical stress has different effects on the forward and reverse reaction kinetics suggests that forward and reverse reactions follow different paths, on average, in the enzyme's conformational space. Comparing the kinetics of the stressed and unstressed enzyme we also show that the maximum speed of the enzyme is comparable to the predictions of the relaxation model of enzyme action, where we use the independently determined dissipation coefficient [Formula: see text] for the enzyme's conformational motion. The present experiments provide a mean to explore enzyme kinetics beyond the static energy landscape picture of transition state theory.

Critical bending torque of DNA is a materials parameter independent of local base sequence.

Short double-stranded DNA molecules exhibit a softening transition under large bending which is quantitatively described by a critical bending torque τ_{c} at which the molecule develops a kink. Through equilibrium measurements of the elastic energy of short (∼10 nm), highly stressed DNA molecules with a nick at the center we determine τ_{c} for different sequences around the nick. We find that τ_{c} is a robust materials parameter essentially independent of sequence. The measurements also show that, at least for nicked DNA, the local structure at the origin of the softening transition is not a single-stranded "bubble."

Mechanical control of Renilla luciferase.

We report experiments where the activity of the enzyme luciferase from Renilla reniformis is controlled through a DNA spring attached to the enzyme. In the wake of previous work on kinases, these results establish that mechanical stress applied through the DNA springs is indeed a general method for the artificial control of enzymes, and for the quantitative study of mechano-chemical coupling in these molecules. We also show proof of concept of the luciferase construct as a sensitive molecular probe, detecting a specific DNA target sequence in an easy, one-step, homogeneous assay, as well as SNP detection without melting curve analysis.

DNA kinks and bubbles: temperature dependence of the elastic energy of sharply bent 10-nm-size DNA molecules.

A 10-nm-long DNA molecule can bend through large angles reversibly. Past the linear regime, its equilibrium nonlinear bending elasticity is governed by a critical bending torque τ(c)≈30pN×nm at which the molecule develops a kink. This nonlinearity has long been attributed to the nucleation of a bubble or melted region in the molecule. Here we measure the temperature dependence of the critical bending torque for nicked DNA, and determine that the entropy associated with the kink in the nonlinear regime is negligible. Thus in the case of nicked DNA the kink is not a bubble, but a compact region deformed beyond a yield strain. We further argue that, with our boundary conditions, the same is likely true for intact DNA. The present measurements confirm that the critical bending torque τ(c) is a materials parameter of DNA mechanics analogous to the bending modulus B≈200pN×nm.

Cracking phase diagram for the dynamics of an enzyme.

We measure the ensemble averaged deformation of an enzyme for an oscillating applied force. From the low frequency divergence of the mechanical susceptibility for the hinge motion of guanylate kinase we obtain a nonequilibrium phase diagram in the frequency-force plane. A phase line separates linear elasticity dynamics from softer (viscoelastic) dynamics. The hinge motion corresponds to crossing this phase line (not to a soft linear elastic mode). The phase line is dramatically shifted in the closed state compared to the open state of the enzyme.

Viscoelastic transition and yield strain of the folded protein.

For proteins, the mechanical properties of the folded state are directly related to function, which generally entails conformational motion. Through sub-Angstrom resolution measurements of the AC mechanical susceptibility of a globular protein we describe a new fundamental materials property of the folded state. For increasing amplitude of the forcing, there is a reversible transition from elastic to viscoelastic response. At fixed frequency, the amplitude of the deformation is piecewise linear in the force, with different slopes in the elastic and viscoelastic regimes. Effectively, the protein softens beyond a yield point defined by this transition. We propose that ligand induced conformational changes generally operate in this viscoelastic regime, and that this is a universal property of the folded state.

Artificial modulation of the gating behavior of a K+ channel in a KvAP-DNA chimera.

We present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. We construct a channel-DNA chimera with the KvAP potassium channel reconstituted in an artificial membrane. The channel is functional and the single channel ion conductivity unperturbed by the presence of the DNA. However, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the DNA and the voltage sensing domain of the protein. Different hybridization states of the chimera DNA thus lead to different response curves of the channel.

Shape of fair weather clouds.

We introduce a model which accounts for the shape of cumulus clouds exclusively in terms of thermal plumes or thermals. The plumes are explicitly represented by a simple potential flow generated by singularities (sources and sinks) and are thus laminar, but with their motion create a field which supports the cloud. We compare this model with actual clouds by means of various shape descriptors including the fractal dimension, and find agreement.

Elasticity of globular proteins measured from the ac susceptibility.

We introduce a new method to measure the elastic constants of globular proteins. Gold nanoparticles, tethered to a gold surface by the protein, are driven by an ac electric field while their displacement is synchronously detected by evanescent wave scattering, yielding the mechanical response function of the macromolecular sample in the frequency domain. We apply the method to measure the stiffening of an enzyme upon binding its substrate.

Controlling proteins through molecular springs.

We argue that the mechanical control of proteins-the notion of controlling chemical reactions and processes by mechanics-is conceptually interesting. We give a brief review of the main accomplishments so far, leading to our present approach of using DNA molecular springs to exert controlled stresses on proteins. Our focus is on the physical principles that underlie both artificial mechanochemical devices and natural mechanisms of allostery.

Elastic energy driven polymerization.

We present a molecular system where polymerization is controlled externally by tuning the elastic energy of the monomers. The elastic energy, provided by a DNA molecular spring, destabilizes the monomer state through a process analogous to domain swapping. This energy can be large (of approximately 10 kT) and thus drive polymerization at relatively low monomer concentrations. The monomer-dimer equilibrium provides a measurement of the elastic energy of the monomer, which in this construction appears limited by kink formation in the DNA molecular spring, in accord with previous theoretical and experimental investigations of the elasticity of sharply bent DNA.

Protein-DNA chimeras: synthesis of two-arm chimeras and non-mechanical effects of the DNA spring.

DNA molecular springs have recently been used to control the activity of enzymes and ribozymes. In this approach, the mechanical stress exerted by the molecular spring alters the enzyme's conformation and thus the enzymatic activity. Here we describe a method alternative to our previous one to attach DNA molecular springs to proteins, where two separate DNA 'arms' are coupled to the protein and subsequently ligated. We report certain non-mechanical effects associated with the DNA spring observed in some chimeras with specific DNA sequences and the nucleotide binding enzyme guanylate kinase. If a ssDNA 'arm' is attached to the protein by one end only, we find that in some cases (depending on the DNA sequence and attachment point on the protein's surface) the unhybridized DNA arm inhibits the enzyme, while hybridization of the DNA arm leads to an apparent activation of the enzyme. One interpretation is that, in these cases, hybridization of the DNA arm removes it from the vicinity of the active site of the enzyme. We show how mechanical and non-mechanical effects of the DNA spring can be distinguished. This is important if one wants to use the protein-DNA chimeras to quantitatively study the response of the enzyme to mechanical perturbations.