Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the Clβ of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher kcat/Km for 1-chlorohexane and a 2-fold higher kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Structure and stability of helices in square-well homopolymers

Recently, it has been demonstrated [Magee et al., Phys. Rev. Lett. 96, 207802 (2006)] that isolated, square-well homopolymers can spontaneously break chiral symmetry and freeze into helical structures at sufficiently low temperatures. This behavior is interesting because the square-well homopolymer is itself achiral. In this work, we use event-driven molecular dynamics, combined with an optimized parallel tempering scheme, to study this polymer model over a wide range of parameters. We examine the conditions where the helix structure is stable and determine how the interaction parameters of the polymer govern the details of the helix structure. The width of the square well (proportional to lambda) is found to control the radius of the helix, which decreases with increasing well width until the polymer forms a coiled sphere for sufficiently large wells. The helices are found to be stable for only a window of molecular weights. If the polymer is too short, the helix will not form. If the polymer is too long, the helix is no longer the minimum energy structure, and other folded structures will form. The size of this window is governed by the chain stiffness, which in this model is a function of the ratio of the monomer size to the bond length. Outside this window, the polymer still freezes into a locked structure at low temperature, however, unless the chain is sufficiently stiff, this structure will not be unique and is similar to a glassy state.Comment: Submitted to Physical Review

Structure, stability and elasticity of DNA nanotube

DNA nanotubes are tubular structures composed of DNA crossover molecules. We present a bottom up approach for construction and characterization of these structures. Various possible topologies of nanotubes are constructed such as 6-helix, 8-helix and tri-tubes with different sequences and lengths. We have used fully atomistic molecular dynamics simulations to study the structure, stability and elasticity of these structures. Several nanosecond long MD simulations give the microscopic details about DNA nanotubes. Based on the structural analysis of simulation data, we show that 6-helix nanotubes are stable and maintain their tubular structure; while 8-helix nanotubes are flattened to stabilize themselves. We also comment on the sequence dependence and effect of overhangs. These structures are approximately four times more rigid having stretch modulus of ~4000 pN compared to the stretch modulus of 1000 pN of DNA double helix molecule of same length and sequence. The stretch moduli of these nanotubes are also three times larger than those of PX/JX crossover DNA molecules which have stretch modulus in the range of 1500-2000 pN. The calculated persistence length is in the range of few microns which is close to the reported experimental results on certain class of the DNA nanotubes.Comment: Published in Physical Chemistry Chemical Physic

Radial Spin Helix in Two-Dimensional Electron Systems with Rashba Spin-Orbit Coupling

We suggest a long-lived spin polarization structure, a radial spin helix, and study its relaxation dynamics. For this purpose, starting with a simple and physically clear consideration of spin transport, we derive a system of equations for spin polarization density and find its general solution in the axially symmetric case. It is demonstrated that the radial spin helix of a certain period relaxes slower than homogeneous spin polarization and plain spin helix. Importantly, the spin polarization at the center of the radial spin helix stays almost unchanged at short times. At longer times, when the initial non-exponential relaxation region ends, the relaxation of the radial spin helix occurs with the same time constant as that describing the relaxation of the plain spin helix.Comment: 9 pages, 7 figure

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

The respiratory syncytial virus nucleoprotein–RNA complex forms a left-handed helical nucleocapsid

Respiratory Syncytial Virus (RSV) is an important human pathogen. Its nucleocapsid (NC), which comprises the negative sense RNA viral genome coated by the viral nucleoprotein N, is a critical assembly that serves as template for both mRNA synthesis and genome replication. We have previously described the X-ray structure of a nucleocapsid-like structure: a decameric ring formed of N-RNA that mimics one turn of the helical NC. In the absence of experimental data we had hypothesized that the NC helix would be right-handed, as the N-N contacts in the ring appeared to more easily adapt to that conformation. We now unambiguously show that the RSV NC is a left-handed helix. We further show that the contacts in the ring can be distorted to maintain key N-N protein interactions in a left-handed helix, and discuss the implications of the resulting atomic model of the helical NC for viral replication and transcription

Weak spin-orbit interactions induce exponentially flat mini-bands in magnetic metals without inversion symmetry

In metallic magnets like MnSi the interplay of two very weak spin-orbit coupling effects can strongly modify the Fermi surface. In the absence of inversion symmetry even a very small Dzyaloshinsky-Moriya interaction of strength delta<<1 distorts a ferromagnetic state into a chiral helix with a long pitch of order 1/delta. We show that additional small spin-orbit coupling terms of order delta in the band structure lead to the formation of exponentially flat minibands with a bandwidth of order exp(-1/sqrt(delta)) parallel to the direction of the helix. These flat minibands cover a rather broad belt of width sqrt(delta) on the Fermi surface where electron motion parallel to the helix practically stops. We argue that these peculiar band-structure effects lead to pronounced features in the anomalous skin effect.Comment: 7 pages, minor corrections, references adde

Magnetic Structure and Magnetization of Helical Antiferromagnets in High Magnetic Fields Perpendicular to the Helix Axis at Zero Temperature

The zero-temperature angles of magnetic moments in a helix or sinusoidal fan confined to the xy plane, with respect to an in-plane magnetic field Hx applied perpendicular to the z axis of a helix or fan, are calculated for commensurate helices and fans with field-independent turn angles kd between moments in adjacent layers of the helix or fan using the classical J0-J1-J2 Heisenberg model. For 0 < kd < $4\pi/9$, first-order transitions from helix to a fan structure occur at fields Ht as previously inferred, where the fan is found to be approximately sinusoidal. However, for $4 \pi/9$ < kd < $\pi$, different behaviors are found depending on the value of kd and these properties vary nonmonotonically with kd. In this kd range, the change from helix to fanlike structure is usually a crossover with no phase transition between them, although first-order and second order transitions are found. We also calculated the average x-axis moment per spin $\mu_{x ave}$ versus Hx for helices and fans with crossovers and phase transitions between them. When smooth helix to fanlike crossovers occur in the range $4\pi/9$ < kd < $\pi$, $\mu_{x ave}$ exhibits an S-shape behavior with increasing Hx. This predicted behavior is consistent with $\mu_{x ave}$(Hx) data previously reported by Sangeetha, et al. [Phys. Rev. B 94, 014422 (2016)] for single-crystal EuCo2P2 possessing a helix ground state with kd = $0.85\pi$. The low-field magnetic susceptibility and the ratio Ht/Hc are calculated analytically or numerically versus kd for helices.Comment: 29 pages, 32 figures, 4 tables; v2: minor corrections, published versio

The Prefusogenic Intermediate of HIV-1 gp41 Contains Exposed C-peptide Regions

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is composed of a complex between the surface subunit gp120, which binds to cellular receptors, and the transmembrane subunit gp41. Upon activation of the envelope glycoprotein by cellular receptors, gp41 undergoes conformational changes that mediate fusion of the viral and cellular membranes. Prior to formation of a fusogenic "trimer-of-hairpins" structure, gp41 transiently adopts a prefusogenic conformation whose structural features are poorly understood. An important approach toward understanding structural conformations of gp41 during HIV-1 entry has been to analyze the structural targets of gp41 inhibitors. We have constructed epitope-tagged versions of 5-Helix, a designed protein that binds to the C-peptide region of gp41 and inhibits HIV-1 membrane fusion. Using these 5-Helix variants, we examined which conformation of gp41 is the target of 5-Helix. We find that although 5-Helix binds poorly to native gp41, it binds strongly to gp41 activated by interaction of the envelope protein with either soluble CD4 or membrane-bound cellular receptors. This preferential interaction with activated gp41 results in the accumulation of 5-Helix on the surface of activated cells. These results strongly suggest that the gp41 prefusogenic intermediate is the target of 5-Helix and that this intermediate has a remarkably "open" structure, with exposed C-peptide regions. These results provide important structural information about this intermediate that should facilitate the development of HIV-1 entry inhibitors and may lead to new vaccine strategies