DNA Local-Flexibility-Dependent Assembly of Phase-Separated Liquid Droplets

Shakya, Anisha,King, John T. Published for the Biophysical Society by the Rocke 2018 Biophysical journal Vol.115 No.10

<P><B>Abstract</B></P> <P>Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid-liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. Comparing the phase behavior of different single-stranded DNA as well as double-stranded DNA (dsDNA) sequences that differ in persistence lengths, we find that DNA local flexibility, not simply charge density, determines the LLPS. Furthermore, in a nucleotide- and DNA-dependent manner, free nucleotide triphosphates promote LLPS of polypeptide-dsDNA complexes that are otherwise prone to precipitation. Under these conditions, dsDNA undergoes a secondary phase separation forming liquid-crystalline subcompartments inside the droplets. These results point toward a role of local DNA flexibility, encoded in the sequence, in the regulation and selectivity of multicomponent LLPS in membraneless intracellular organization.</P>

Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A_2A Adenosine Receptor

Lee, Y.,Kim, S.,Choi, S.,Hyeon, C. Published for the Biophysical Society by the Rocke 2016 Biophysical journal Vol.111 No.6

<P>Water molecules inside a G-protein coupled receptor (GPCR) have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal water molecules in GPCR activity, we studied the A(2A) adenosine receptor using microsecond molecular-dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times more slowly than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from similar to O(10(2)) ps to similar to O(10(2)) ns. Especially, water molecules, exhibiting ultraslow relaxation (similar to O(10(2)) ns) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow water molecules in the activation of GPCRs.</P>

Ligand-Mediated Folding of the OmpA Periplasmic Domain from <i>Acinetobacter baumannii</i>

Mushtaq, Ameeq Ul,Park, Jeong Soon,Bae, Sung-Hun,Kim, Hye-Yeon,Yeo, Kwon Joo,Hwang, Eunha,Lee, Ki Yong,Jee, Jun-Goo,Cheong, Hae-Kap,Jeon, Young Ho Published for the Biophysical Society by the Rocke 2017 Biophysical journal Vol.112 No.10

<P>The periplasmic domain of OmpA from Acinetobacter baumannii (AbOmpA-PD) binds to diaminopimelate and anchors the outer membrane to the peptidoglycan layer in the cell wall. Although the crystal structure of AbOmpA-PD with its ligands has been reported, the mechanism of ligand-mediated folding of AbOmpA remains elusive. Here, we report that in vitro refolded apo-AbOmpA-PD in the absence of ligand exists as a mixture of two partially folded forms in solution: mostly unfolded (apo-state I) and hololike (apo-state II) states. Binding of the diaminopimelate or glycine ligand induced complete folding of AbOmpA-PD. The apo-state I was highly flexible and contained some secondary structural elements, whereas the apo-state II closely resembled the holo-state in terms of both structure and backbone dynamics, except for the ligand-binding region. N-15-relaxation-dispersion analyses for apo-state II revealed substantial motion on a millisecond timescale of residues in the H3 helix near the ligand-binding site, with this motion disappearing upon ligand binding. These results provide an insight into the ligand-mediated folding mechanism of AbOmpA-PD in solution.</P>

Symmetry-Constrained Normal Mode Analysis of the Bacterial Flagellar Motor

Ki Choi, M.,Jo, S.,Ki, M. Published for the Biophysical Society by the Rocke 2016 Biophysical journal Vol.110 No.3

Effects of Dimethyl Sulfoxide on Surface Water near Phospholipid Bilayers

Lee, Yuno,Pincus, Philip A.,Hyeon, Changbong Published for the Biophysical Society by the Rocke 2016 Biophysical journal Vol.111 No.11

<P>Despite much effort to probe the properties of dimethyl sulfoxide (DMSO) solution, the effects of DMSO on water, especially near plasma membrane surfaces, still remain elusive. By performing molecular dynamics simulations at varying DMSO concentrations (X-DMSO), we study how DMSO affects structural and dynamical properties of water in the vicinity of phospholipid bilayers. As proposed by a number of experiments, our simulations confirm that DMSO induces dehydration from bilayer surfaces and disrupts the H-bond structure of water. However, DMSO-enhanced water diffusivity at solvent-bilayer interfaces, an intriguing discovery reported by a spin-label measurement, is not confirmed in our simulations. To resolve this discrepancy, we examine the location of the spin label (Tempo) relative to the solvent-bilayer interface. In accord with the evidence in the literature, our simulations, which explicitly model Tempo-phosphatidylcholine, find that the Tempo moiety is equilibrated at similar to 8-10 angstrom below the bilayer surface. Furthermore, the DMSO-enhanced surface-water diffusion is confirmed only when water diffusion is analyzed around the Tempo moiety that is immersed below the bilayer surface, which implies that the experimentally detected signal of water using Tempo stems from the interior of bilayers, not from the interface. Our analysis finds that the increase of water diffusion below the bilayer surface is coupled to the increase of area per lipid with an increasing XDMSO (less than or similar to 10 mol %). Underscoring the hydrophobic nature of the Tempo moiety, our study calls for careful re-evaluation of the use of Tempo in measurements on lipid bilayer surfaces.</P>

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca²⁺ Channels

Kim, D.I.,Kang, M.,Kim, S.,Lee, J.,Park, Y.,Chang, I.,Suh, B.C. Published for the Biophysical Society by the Rocke 2015 Biophysical journal Vol.109 No.5

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (Ca<SUB>V</SUB>) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP<SUB>2</SUB>). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of Ca<SUB>V</SUB> channels by PIP<SUB>2</SUB>. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP<SUB>2</SUB> sensitivity of Ca<SUB>V</SUB>2.2 and Ca<SUB>V</SUB>1.3 channels by ~3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.

Annealed Random Copolymer Model of the B-Z Transition in DNA: Torsional Responses

Kwon, A.Y.,Lee, N.K.,Hong, S.C.,Fierling, J.,Johner, A. Published for the Biophysical Society by the Rocke 2015 Biophysical journal Vol.108 No.10

Both in vivo and in vitro, specific sequences in double-stranded DNA can adopt the left-handed Z-form when underwound. Recently, the B-Z transition of DNA has been studied in detail in magnetic tweezers experiments by several groups. We present a theoretical description of this transition, based on an annealed random copolymer model. The transition of a switchable sequence is discussed as a function of energetic and geometric parameters of the B- and Z-forms, of the applied boundary conditions, and of the characteristics of the B-Z interface. We address a possible torsional softening upon the B-Z transition. The model can be also applied to other biofilaments with annealed torsional/flexural degrees of freedom.

Gestation of a Glu Plasminogen Supra Fold via Molecular Dynamics Simulation

Kim, H.,Kim, H.J.,Llinas, M. Published for the Biophysical Society by the Rocke 2015 Biophysical journal Vol.108 No.2

Contact Statistics Highlight Distinct Organizing Principles of Proteins and RNA

Liu, L.,Hyeon, C. Published for the Biophysical Society by the Rocke 2016 Biophysical journal Vol.110 No.11

<P>Although both RNA and proteins have densely packed native structures, chain organizations of these two biopolymers are fundamentally different. Motivated by the recent discoveries in chromatin folding that interphase chromosomes have territorial organization with signatures pointing to metastability, we analyzed the biomolecular structures deposited in the Protein Data Bank and found that the intrachain contact probabilities, P(s) as a function of the arc length s, decay in power-law similar to s(-gamma) over the intermediate range of s, 10 less than or similar to s less than or similar to 110. We found that the contact probability scaling exponent is gamma approximate to 1.11 for large RNA (N > 110), gamma approximate to 1.41 for small-sized RNA (N < 110), and gamma approximate to 1.65 for proteins. Given that Gaussian statistics is expected for a fully equilibrated chain in polymer melts, the deviation of gamma-value from gamma = 1.5 for the subchains of large RNA in the native state suggests that the chain configuration of RNA is not fully equilibrated. It is visually clear that folded structures of large-sized RNA (N greater than or similar to 110) adopt crumpled structures, partitioned into modular multidomains assembled by proximal sequences along the chain, whereas the polypeptide chain of folded proteins looks better mixed with the rest of the structure. Our finding of gamma approximate to 1 for large RNA might be an ineluctable consequence of the hierarchical ordering of the secondary to tertiary elements in the folding process.</P>

Ring-Shaped Replicative Helicase Encircles Double-Stranded DNA during Unwinding

Lee, Mina,Joo, Sihwa,Hwan Ha, Tai Published for the Biophysical Society by the Rocke 2020 Biophysical journal Vol. No.