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Targeted protein degradation (TPD) has emerged as a powerful approach in drug discovery for removing (rather than inhibiting) proteins implicated in diseases. A key step in this approach is the formation of an induced proximity complex, where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) assessment of ubiquitination propensity via the full Cullin Ring Ligase (CRL) macromolecular assembly. The novel approach presented here combines experimental biophysical data—in this case hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues)—with all-atom explicit solvent molecular dynamics (MD) simulations aided by enhanced sampling techniques to predict structural ensembles of ternary complexes at atomic resolution. We present results demonstrating the efficiency, accuracy, and reliability of our approach to predict ternary structure ensembles using the bromodomain of SMARCA2 (SMARCA2BD) with the E3 ligase VHL as the system of interest. The simulations reproduce X-ray crystal structures – including prospective simulations validated on a new structure that we determined in this work (PDB ID: 7S4E) – with root mean square deviations (RMSD) of 1.1 to 1.6 Å. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex within a broad energy basin. To further characterize the structural ensemble, we used snapshots from the aforementioned simulations as seeds for Hamiltonian replica exchange molecular dynamics (HREMD) simulations, and then perform 7.1 milliseconds of aggregate simulation time using Folding@home. The resulting free energy surface identifies the crystal structure conformation within a broad low-energy basin and the dynamic ensemble is consistent with solution-phase biophysical experimental data (HDX-MS and small-angle x-ray scattering, SAXS). Finally, we graft structures from the ternary complexes onto the full CRL and perform enhanced sampling simulations, where we find that differences in degradation efficiency can be explained by the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. Several of the top predicted ubiquitinated lysine residues are validated prospectively through a ubiquitin mapping proteomics experiment.

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes, play a major role in cell signaling, and are associated with human diseases. To understand IDP function it is critical to determine their configurational ensemble, i.e., the collection of 3-dimensional structures they adopt, and this remains an immense challenge in structural biology. Attempts to determine this ensemble computationally have been hitherto hampered by the necessity of reweighting molecular dynamics (MD) results or biasing simulation in order to match ensemble-averaged experimental observables, operations that reduce the precision of the generated model because different structural ensembles may yield the same experimental observable. Here, by employing enhanced sampling MD we reproduce the experimental small-angle neutron and X-ray scattering profiles and the NMR chemical shifts of the disordered N terminal (SH4UD) of c-Src kinase without reweighting or constraining the simulations. The unbiased simulation results reveal a weakly funneled and rugged free energy landscape of SH4UD, which gives rise to a heterogeneous ensemble of structures that cannot be described by simple polymer theory. SH4UD adopts transient helices, which are found away from known phosphorylation sites and could play a key role in the stabilization of structural regions necessary for phosphorylation. Our findings indicate that adequately sampled molecular simulations can be performed to provide accurate physical models of flexible biosystems, thus rationalizing their biological function.

Substituted xylans play an important role in the structure and mechanics of the primary cell wall of plants. Arabinoxylans (AX) consist of a xylose backbone substituted with arabinose, while glucuronoarabinoxylans (GAX) also contain glucuronic acid substitutions and ferulic acid esters on some of the arabinoses. We provide a molecular-level description on the dependence of xylan conformational, self-aggregation properties and binding to cellulose on the degree of arabinose substitution. Molecular dynamics simulations reveal fully solubilized xylans with a low degree of arabinose substitution (lsAX) to be stiffer than their highly substituted (hsAX) counterparts. Small-angle neutron scattering experiments indicate that both wild-type hsAX and debranched lsAX form macromolecular networks that are penetrated by water. In those networks, lsAX are more folded and entangled than hsAX chains. Increased conformational entropy upon network formation for hsAX contributes to AX loss of solubility upon debranching. Furthermore, simulations show the intermolecular contacts to cellulose are not affected by arabinose substitution (within the margin of error). Ferulic acid is the GAX moiety found here to bind to cellulose most strongly, suggesting it may play an anchoring role to strengthen GAX-cellulose interactions. The above results suggest highly substituted GAX acts as a spacer, keeping cellulose microfibrils apart, whereas low substitution GAX is more localized in plant cell walls and promotes cellulose bundling.

We have investigated the effect of molecular shape and charge asymmetry on the translation dynamics of confined hydrocarbon molecules having different shapes but similar kinetic diameters, inside ZSM-5 pores using molecular dynamics simulations. The mean square displacement of propane, acetonitrile, acetaldehyde, and acetone in ZSM-5 exhibit two different regimes - ballistic and diffusive/sub-diffusive. All the molecules except propane exhibit sub-diffusive motion at time scales greater than 1 ps. The intermediate scattering functions reveal that there is a considerable rotational- translational coupling in the motion of all the molecules, due to the strong geometrical restriction imposed by ZSM-5. Overall the difference in shape and asymmetry in charge imposes severe restriction inside the ZSM-5 channels for all the molecules to different extents. Further, the behavior of molecules confined in ZSM-5 in the present study, quantified wherever possible, is compared to their behavior in bulk or in other porous media reported in literature., 10 Pages, 10 Figures

Molecular dynamics (MD) simulation is widely used to complement ensemble-averaged experiments of intrinsically disordered proteins (IDPs). However, MD often suffers from limitations of inaccuracy. Here, we show that enhancing the sampling using Hamiltonian replica-exchange MD (HREMD) led to unbiased and accurate ensembles, reproducing small-angle scattering and NMR chemical shift experiments, for three IDPs of varying sequence properties using two recently optimized force fields, indicating the general applicability of HREMD for IDPs. We further demonstrate that, unlike HREMD, standard MD can reproduce experimental NMR chemical shifts, but not small-angle scattering data, suggesting chemical shifts are insufficient for testing the validity of IDP ensembles. Surprisingly, we reveal that despite differences in their sequence, the inter-chain statistics of all three IDPs are similar for short contour lengths (< 10 residues). The results suggest that the major hurdle of generating an accurate unbiased ensemble for IDPs has now been largely overcome., Shrestha et al. show that enhancing the sampling using Hamiltonian replica exchange molecular simulation (HREMD) leads to accurate unbiased ensembles of intrinsically disordered proteins. They find that standard molecular simulation cannot reproduce small-angle scattering data as well as HREMD, highlighting the utility of the suggested approach.

The mesophilic inorganic pyrophosphatase from Escherichia coli (EcPPase) retains function at 353 K, the physiological temperature of hyperthermophilic Thermococcus thioreducens, whereas the homolog protein (TtPPase) from this hyperthermophilic organism cannot function at room temperature. To explain this asymmetric behavior, we examined structural and dynamical properties of the two proteins using molecular dynamics simulations. The global flexibility of TtPPase is significantly higher than its mesophilic homolog at all tested temperature/pressure conditions. However, at 353 K, EcPPase reduces its solvent-exposed surface area and increases subunit compaction while maintaining flexibility in its catalytic pocket. In contrast, TtPPase lacks this adaptability and has increased rigidity and reduced protein/water interactions in its catalytic pocket at room temperature, providing a plausible explanation for its inactivity near room temperature.

The mesophilic inorganic pyrophosphatase fromEscherichia coli(EcPPase) retains function at 353 K, the physiological temperature of hyperthermophilicThermoccoccus thioreducens, whereas, the homolog protein from the hyperthermophilic organism (TtPPase) cannot function at room temperature. To explain this asymmetric behavior, we examined structural and dynamical properties of the two proteins using molecular dynamics simulations. The global flexibility ofTtPPase is significantly higher than its mesophilic homolog at all tested temperature/pressure conditions. However, at 353 K,EcPPase reduces its solvent-exposed surface area and increases subunit compaction while maintaining flexibility in its catalytic pocket. In contrast,TtPPase lacks this adaptability and has increased rigidity and reduced protein:water interactions in its catalytic pocket at room temperature, providing a plausible explanation for its inactivity near room temperature.

Orientational structure and dynamics of molecules is known to be affected by confinement in space comparable in size to the molecule itself. ZSM-5 with porous channels of ≈ 0.55 nm is such a porous medium, which offers a strict spatial confinement on low molecular weight hydrocarbons. An important factor that determines these properties is the shape of the confined molecules. We employed molecular dynamics simulation to study the orientational structure and dynamics of four molecules that differ in shape but have similar kinetic diameters and moments of inertia, confined in ZSM-5. The effect of molecular shape on the orientational structure and dynamics of propane, acetonitrile, acetaldehyde and acetone in ZSM-5 is studied by means of probing the differences in the orientational distribution of molecules in the ZSM-5 channels, and extracting time scales of the decay of correlation functions related to rotational motion. Orientational correlation functions of all the four molecules exhibit two regimes of rotational motion. While the short time regime represents free rotation of the molecules before they collide with the pore walls, the long time orientational jumps driven by inter-channel migrations give rise to a very slow varying second regime. Of the molecules studied, orientational structure and dynamics of propane is found to be least affected by confinement under ZSM-5, whereas charge and shape asymmetry of other molecules makes their interchannel migration-driven rotation slow. The time scales involved in the rotational motion for the molecules studied are compared with similar studies reported in literature. This study reveals the important role that molecular shape plays in the behavior of confined molecules.

Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond-nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differences can be attributed to the influence of the covalently bound retinal ligand. Furthermore, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.

Introductions: Although Closed Reduction and Percutaneous Pinning is the gold standard of treatment for Supracondylar fractures (SC) in children, reduction is not always easy. Minimally Invasive, Closed Reduction and Percutaneous Pinning (MI-CRPP) reduces the soft tissue trauma and provides easy reduction. We have reviewed the success rate of minimally invasive reduction technique and its outcome. Methods: We reviewed the charts of 155 children (97 male, 58 female) age ranging from 2 to 14 years with SC fractures of the humerus who were operated with minimally invasive closed reduction and precutaneous pinning from November 2008 to June 2014 at Patan Hospital and Om Hospital. They were followed up for a mean of eight (4 to 24) weeks. The K-wires were removed at 4 to 6 (average 4.28) weeks. Results: Male children were affected more than female with the ratio being 97 to 58. Right side was affected more than left (ratio 89 to 66). Post-operatively, there were six (3.87%) ulnar nerve injury and eight (5.16%) patients came with superficial pin tract infection. One hundred and thirteen (72.9%) had excellent, 35 (22.58%) good, five (3.23%) fair and two (1.3%) poor results at the eight week follow-up which was improved to 144 (92.9%) excellent, seven (4.5%) good, three (1.9%) fair and one (0.65%) poor results at the 14 week follow-up. Conclusions: Closed reduction of supracondylar fractures of the humerus in children with minimally invasive technique prior to K-wire fixation is a relatively simple, safe and effective method of achieving satisfactory reduction and good functional outcome. Keywords: cubitus varus deformity, K wire fixation,minimally invasive closed reduction and precutaneous pinning, supracondylar fractures

Knowledge of the activation principles for G-protein-coupled receptors (GPCRs) is critical to development of new pharmaceuticals. Rhodopsin is the archetype for the largest GPCR family, yet the changes in protein dynamics that trigger signaling are not fully understood. Here we show that rhodopsin can be investigated by small-angle neutron scattering (SANS) in fully protiated detergent micelles under contrast matching to resolve light-induced changes in the protein structure. In SANS studies of membrane proteins, the zwitterionic detergent [(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS) is advantageous because of the low contrast difference between the hydrophobic core and hydrophilic head groups as compared with alkyl glycoside detergents. Combining SANS results with quasielastic neutron scattering reveals how changes in volumetric protein shape are coupled (slaved) to the aqueous solvent. Upon light exposure, rhodopsin is swollen by the penetration of water into the protein core, allowing interactions with effector proteins in the visual signaling mechanism.

Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from a hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is up to 100 MPa (1 kbar). It has attracted great interest in biophysical research because of its high activity under extreme conditions in the seabed. In this study, we use the quasielastic neutron scattering (QENS) technique to investigate the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range. The β-relaxation dynamics of proteins was studied in the time ranges from 2 to 25 ps, and from 100 ps to 2 ns, using two spectrometers. Our results indicate that, under a pressure of 100 MPa, close to that of the native environment deep under the sea, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), at all measured temperatures, opposite to what we observed previously under ambient pressure. This contradictory observation provides evidence that the protein energy landscape is distorted by high pressure, which is significantly different for hyperthermophilic (IPPase) and mesophilic (HEWL) proteins. We further derive from our observations a schematic denaturation phase diagram together with energy landscapes for the two very different proteins, which can be used as a general picture to understand the dynamical properties of thermophilic proteins under pressure.

In this article, we elucidate the protein activity from the perspective of protein softness and flexibility by studying the collective phonon-like excitations in a globular protein, human serum albumin (HSA), and taking advantage of the state-of-the-art inelastic X-ray scattering (IXS) technique. Such excitations demonstrate that the protein becomes softer upon thermal denaturation due to disruption of weak noncovalent bonds. On the other hand, no significant change in the local excitations is detected in ligand- (drugs) bound HSA compared to the ligand-free HSA. Our results clearly suggest that the protein conformational flexibility and rigidity are balanced by the native protein structure for biological activity.

Protein fluctuations are the key for activation of G-protein-coupled receptors (GPCRs) such as rhodopsin. X-ray crystallography reveals useful structural information about the intermediates in the photoactivation process; however, knowledge of protein dynamical changes is pivotal for understanding the activation mechanism of GPCRs. We hypothesized that rhodopsin activation leads to multiple activated states in accord with an ensemble-activation mechanism (EAM) [1]. Neutron scattering provides us with non-invasive techniques to study both structural and dynamical transitions associated with the function of physiologically important proteins such as rhodopsin. Here we describe a combined small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS) approach to investigate the structural fluctuations associated with rhodopsin activation. The samples were prepared by purifying rhodopsin in detergents such as CHAPS from rhodopsin disk membranes. In SANS the intensity of the neutrons scattered is measured as a function of momentum transfer (Q). The experiments conducted on detergent contrast-matched conditions allowed us to probe the light-induced structural changes in rhodopsin exclusively in protein-detergent micelles. The SANS study unveiled a volumetric expansion of the protein upon photoactivation of rhodopsin. In QENS, the energy spectrum of the neutron scattered as a function of transfer-energy (ω) is measured for partially hydrated (h ≈ 0.27) rhodopsin samples. The QENS data in conjunction with the mode-coupling theory (MCT) revealed that the β-fluctuations in ligand-free opsin are substantially slower than in the dark-state rhodopsin, which indicates an increase in protein flexibility upon rhodopsin photoactivation. The volumetric expansion (from SANS experiments) and increased protein flexibility (from QENS experiments) are consistent with an increase in the number of configurations upon rhodopsin photoactivation as previously suggested by an EAM. Hence, neutron scattering provides insights into protein fluctuations crucial for GPCR activation. [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268.

Understanding G-protein-coupled receptor (GPCR) activation plays a crucial role in the development of novel improved molecular drugs. During photoactivation, the retinal chromophore of the visual GPCR rhodopsin isomerizes from the 11-cis conformation to the all-trans conformation, yielding an equilibrium between inactive Meta-I and active Meta-II states [1]. The principal goals of this work are to address whether the activation of rhodopsin leads to a single state or a conformational ensemble, and how the protein organizational structure changes with the detergent environment. We used small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) to answer the above questions. Both SANS and SAXS are powerful techniques to study the macromolecular structures in solution within the length scale from angstroms to several nanometers. In our experiments, rhodopsin is solubilized in CHAPS detergent, which favors the inactive Meta-I state. By contrast, dodecylmaltoside (DDM) detergent stabilizes the active Meta-II state [2]. Notably SANS with contrast-variation enables the separate study of the protein structure within the detergent assembly [3], and suggests a looser structure of rhodopsin in DDM versus CHAPS micelles. Such results are consistent with the SAXS data fitted by either a core-shell ellipsoid or core-shell cylindrical model, describing a monolayer of detergent molecules surrounding the rhodopsin molecule. Moreover, the SAXS experiments with different rhodopsin to detergent ratios delineate the role of the detergent in stabilization of the protein in solution. Our combined approach of SANS and SAXS studies reveals the protein structural changes associated with GPCR activation in the case of visual rhodopsin.[1] A. V. Struts et al. (2011) PNAS 108, 8263-8268.[2] A. V. Struts et al. (2014) Meth. Mol. Biol. in press.[3] R. K. Le et al. (2014) Arch. Biochem. Biophys. 550-551, 50-57.

Detecting the phonon dispersion relations in proteins is essential for understanding the intra-protein dynamical behavior. Such study has been attempted by X-ray in recent years [1,2]. However, for such detections, neutrons have significant advantages in resolution and time-efficiency compare to X-rays. Traditionally the collective motions of atoms in protein molecules are hard to detect using neutrons, because of high incoherent scattering background from intrinsic hydrogen atoms in the protein molecules. The recent availability of a fully deuterated green fluorescent protein (GFP) synthesized by the Bio-Deuteration Lab at ORNL opens new possibilities to probe collective excitations in proteins using inelastic neutron scattering [3]. Using a direct time-of-flight Fermi chopper neutron spectrometer, we obtained a full map of the milli-eV phonon-like excitations in the fully deuterated protein. The Q range of the observed excitations corresponds to the length scale of about 2.5 to 3 A, which is close to the length scales of the secondary structures of proteins (4-5 A) and reflects the collective intra-protein motions. Our results show that hydration of GFP seems to harden, not soften, the collective motions, which is counterintuitive, but in agreement with the observations by previous neutron scattering experiments [4].[1] D. Liu, X.-Q. Chu, M. Lagi, Y. Zhang, E. Fratini, P. Baglioni, A. Alatas, A. Said, E. Alp, and S.-H. Chen, Phys. Rev. Lett. 101, 135501 (2008).[2] M. Li, X.-Q. Chu, E. Fratini, P. Baglioni, A. Alatas, E. Alp, and S.-H. Chen, Soft Matter 7, 9848-9853 (2011).[3] X.-Q. Chu, U. Shrestha, H. O'Neill, Q. Zhang, A. I. Kolesnikov, and E. Mamontov, "Mapping meV excitations in a fully deuterated green fluorescent protein by time-of-flight neutron scattering", to be submitted.[4] X.-Q. Chu, et. al, J. Phys. Chem. B 113, 5001 (2009).

Protein dynamics or protein motions are believed to ultimately govern the biological function and activities of protein. Quasielastic neutron scattering (QENS) technique has been proven to be an exceptional tool to study dynamics of proteins in the time scale of picosecond (ps) to nanosecond (ns) [1, 2]. In this study, we use QENS to investigate how a large oligomeric protein, Inorganic Pyrophosphatase (IPPase) from Thermococcus thioreducens with quaternary structural complexity, have distinguishable dynamic characteristics compared to those of the small simple monomeric model protein, lysozyme. IPPase derived from thermostable microorganisms is of extreme interest for biophysical studies because of their inherent chemical and thermal stability and high temperature activity. Two QENS instruments, a backscattering spectrometer (BASIS) and a disk chopper spectrometer (DCS) are used in probing the protein dynamics in different time ranges from 1 ps to 1 ns at different temperatures. In addition, the DCS experiment was performed under the pressure of 1000 bar, mimicking the natural living conditions of IPPase. Our results reveal that the dynamics of IPPase is slower than that of lysozyme in the time range of 10 ps to 0.5 ns [1] while it is faster in the time range of 1 ps to 30 ps. These results are consistent between two instruments and such dynamic behaviors in proteins are believed to be contributed by the rotational motion of the side methyl groups [3]. Distinguishable dynamical behavior found between two proteins reveals local flexibility and conformational substates unique to oligomeric structures. Our results greatly help understanding the relation between protein dynamics and their biological functions.[1] X.-Q. Chu, et al, JPCB. 116, 9917 (2012).[2] X.-Q. Chu, et al, Soft Matter 6, 2623 (2010).[3] X.-Q. Chu, et al, JPCL. 4, 936 (2013).

G-protein-coupled receptors (GPCRs) represent the largest family of proteins in the human genome and comprise about 50% of current molecular drug targets. Rhodopsin is the GPCR involved in visual light perception and occurs naturally in a membrane lipid environment. Rhodopsin photoactivation yields cis-trans isomerization of retinal giving an equilibrium between inactive Meta-I and active Meta-II states. Here we address the question: does photoactivation lead to a single Meta-II conformation or is there an ensemble of substates described by an ensemble-activation mechanism (EAM) [1]? In this context small-angle neutron scattering (SANS) probes rhodopsin-detergent and rhodopsin-lipid complexes through measurement of the intensity of the neutrons scattered as a function of scattering vector I(q). Contrast variation enables us to highlight individual components of a multi-component system without isotopic labeling of the sample. Upon photoactivation, the Meta-I state was stabilized in CHAPS-solubilized rhodopsin, while Meta-II was trapped in DDM-solubilized rhodopsin. The ligand-free apoprotein opsin was obtained by photobleaching rhodopsin in the presence of hydroxylamine. The SANS spectra for the above rhodopsin substates were acquired from 80% D2O solutions and at contrast-matching points for both DDM and CHAPS samples. The data collected in 80% D2O samples provide structural information for both protein and detergent, while the data collected at contrast-matching points give information for the protein structure exclusively. Our experiments demonstrate that for detergent-solubilized rhodopsin, SANS with contrast variation can detect structural differences between the rhodopsin dark-state, Meta-I, Meta-II, and ligand-free opsin states. Dark-state rhodopsin is more conformationally flexible (less-compact) in DDM micelles compared to the CHAPS, which is consistent with an ensemble of activated Meta-II states. Furthermore, the time-dependent structural transitions between Meta-I and Meta-II as observed by time-resolved SANS will be crucial to understanding the ensemble-based activation. [1]A.V. Struts et al. (2011) NSMB18, 392-394.

Life is traditionally seen as being driven by energy from the sun, however deep sea organisms have no access to sunlight, so they depend on nutrients found in the dusty chemical deposits and hydrothermal fluids around the hydrothermal vent zones, where they live. In this study, we use neutron spin-echo spectroscopy (NSE) to measure the inter-domain motions of the inorganic pyrophosphatase (IPPase) enzyme derived from thermostable microorganisms. IPPase is of extreme interest for biophysical studies because of their inherent chemical and thermal stability and high temperature activity. It has a hexameric quaternary structure with a molecular mass of approximately 120kDa (each subunit is about 20kDa molecular weight), which is a large oligomeric molecular structure. Study of the slow inter-domain motions that occur in the protein is the key to understand why IPPase can perform catalytic activity at much higher temperature than normal enzymes, thus can adapt to the extreme environment present at the seabed [1-3]. NSE spectroscopy is able to probe these slow inter-domain motions directly in the time-domain, as has already been established in other studies[4,5]. The length and timescale of NSE are right in the ranges from sub-Angstrom and picoseconds to nanometers and several tens of nanoseconds and beyond. Distinguishable dynamical behavior found between two proteins reveals local flexibility and conformational substates unique to oligomeric structures. Our results greatly help understanding the relation between protein dynamics and their biological functions.[1] X.-Q. Chu, et. al, JPCB 116, 9917 (2012).[2] X.-Q. Chu, et. al, Soft Matter 6, 2623(2010).[3] X.-Q. Chu, et. al, JPCL 4, 936 (2013).[4] R. Biehl, et. al, Soft Matter 7, 1299 (2011).[5] N. Smolin, et. al, Biophys. J. 102, 1108 (2012).