Long-range Structural Changes Research Articles

Single-cell landscape of nuclear configuration and gene expression during stem cell differentiation and X inactivation

BackgroundMammalian development is associated with extensive changes in gene expression, chromatin accessibility, and nuclear structure. Here, we follow such changes associated with mouse embryonic stem cell differentiation and X inactivation by integrating, for the first time, allele-specific data from these three modalities obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C.ResultsAllele-specific contact decay profiles obtained by single-cell Hi-C clearly show that the inactive X chromosome has a unique profile in differentiated cells that have undergone X inactivation. Loss of this inactive X-specific structure at mitosis is followed by its reappearance during the cell cycle, suggesting a “bookmark” mechanism. Differentiation of embryonic stem cells to follow the onset of X inactivation is associated with changes in contact decay profiles that occur in parallel on both the X chromosomes and autosomes. Single-cell RNA-seq and ATAC-seq show evidence of a delay in female versus male cells, due to the presence of two active X chromosomes at early stages of differentiation. The onset of the inactive X-specific structure in single cells occurs later than gene silencing, consistent with the idea that chromatin compaction is a late event of X inactivation. Single-cell Hi-C highlights evidence of discrete changes in nuclear structure characterized by the acquisition of very long-range contacts throughout the nucleus. Novel computational approaches allow for the effective alignment of single-cell gene expression, chromatin accessibility, and 3D chromosome structure.ConclusionsBased on trajectory analyses, three distinct nuclear structure states are detected reflecting discrete and profound simultaneous changes not only to the structure of the X chromosomes, but also to that of autosomes during differentiation. Our study reveals that long-range structural changes to chromosomes appear as discrete events, unlike progressive changes in gene expression and chromatin accessibility.

Structural Analysis of Anti-Hapten Antibodies to Identify Long-Range Structural Movements Induced by Hapten Binding.

Antibodies are well known for their high specificity that has enabled them to be of significant use in both therapeutic and diagnostic applications. Antibodies can recognize different antigens, including proteins, carbohydrates, peptides, nucleic acids, lipids, and small molecular weight haptens that are abundantly available as hormones, pharmaceuticals, and pesticides. Here we focus on a structural analysis of hapten-antibody couples and identify potential structural movements originating from the hapten binding by comparison with unbound antibody, utilizing 40 crystal structures from the Protein Data Bank. Our analysis reveals three binding surface trends; S1 where a pocket forms to accommodate the hapten, S2 where a pocket is removed when the hapten binds, and S3 where no pockets changes are found. S1 and S2 are expected for induced-fit binding, whereas S3 indicates that a pre-existing population of optimal binding antibody conformation exists. The structural analysis reveals four classifications of structural reorganization, some of which correlate to S2 but not to the other binding surface changes. These observations demonstrate the complexity of the antibody-antigen interaction, where structural changes can be restricted to the binding sites, or extend through the constant domains to propagate structural changes. This highlights the importance of structural analysis to ensure successful and compatible transformation of small antibody fragments at the early discovery stage into full antibodies during the subsequent development stages, where long-range structural changes are required for an Fc effector response.

Essential role of accessory subunit LYRM6 in the mechanism of mitochondrial complex I

Respiratory complex I catalyzes electron transfer from NADH to ubiquinone (Q) coupled to vectorial proton translocation across the inner mitochondrial membrane. Despite recent progress in structure determination of this very large membrane protein complex, the coupling mechanism is a matter of ongoing debate and the function of accessory subunits surrounding the canonical core subunits is essentially unknown. Concerted rearrangements within a cluster of conserved loops of central subunits NDUFS2 (β1-β2S2 loop), ND1 (TMH5-6ND1 loop) and ND3 (TMH1-2ND3 loop) were suggested to be critical for its proton pumping mechanism. Here, we show that stabilization of the TMH1-2ND3 loop by accessory subunit LYRM6 (NDUFA6) is pivotal for energy conversion by mitochondrial complex I. We determined the high-resolution structure of inactive mutant F89ALYRM6 of eukaryotic complex I from the yeast Yarrowia lipolytica and found long-range structural changes affecting the entire loop cluster. In atomistic molecular dynamics simulations of the mutant, we observed conformational transitions in the loop cluster that disrupted a putative pathway for delivery of substrate protons required in Q redox chemistry. Our results elucidate in detail the essential role of accessory subunit LYRM6 for the function of eukaryotic complex I and offer clues on its redox-linked proton pumping mechanism.

Role of electronic and magnetic interactions in defect formation and anomalous diffusion in δ-Pu

Previous experimental work has shown self-irradiation in Pu solids produces point defect populations that correlate with increases in local disorder, long-range structural changes, induced magnetic moments, and other thermo-physical property changes. Thermally activated kinetic processes drive these defects to diffuse and interact toward either damage evolution or lattice recovery. Using DFT and cNEB, as implemented in VASP, barriers for mono-vacancy and split-interstitial diffusion and Frenkel pair recombination were calculated in fcc δ-Pu. The results indicate the migration barrier of a monoclinic mono-vacancy is lower when compared to the migration barrier of a tetragonal split-interstitial in δ-Pu, contrary to typical fcc metal point defect migration. This fundamentally different diffusion mechanism is a result of local symmetry breaking induced by electronic and magnetic interactions leading to the development of Pu–Pu short bonds (<3.0 Å) within a many-atom complex defect forming and migrating. The migration of the monoclinic mono-vacancy maintains short bonds with anti-parallel spins throughout the transition; whereas, during the migration transition state for the tetragonal split-interstitial, formation of short bonds with parallel spins and a spin-flip of the migrating Pu interstitial occurs. The associated energy cost is reflected in an increase in the migration barrier energy. Frenkel pair recombination is not spontaneous at 0K, but correlates with magnetic moment interactions, leading to an energy barrier for recombination. From these results, it is concluded that migration of defects in unalloyed δ-Pu are highly dependent on the electronic and magnetic interactions that induce associated low-symmetry structures and consequently influence the diffusional properties. Typical fcc defect diffusion mechanisms do not apply to the monoclinic mono-vacancy and tetragonal split-interstitial in the complex 5f δ-Pu system suggesting that the experimental observation of radiation damage induced localized magnetic moments and anomalous diffusion properties measured in δ-Pu could be understood in terms of defect kinetics and interactions.

Cation Disorder and Lithium Insertion Mechanism of Wadsley-Roth Crystallographic Shear Phases from First Principles.

Wadsley-Roth crystallographic shear phases form a family of compounds that have attracted attention due to their excellent performance as lithium-ion battery electrodes. The complex crystallographic structure of these materials poses a challenge for first-principles computational modeling and hinders the understanding of their structural, electronic and dynamic properties. In this article, we study three different niobium-tungsten oxide crystallographic shear phases (Nb12WO33, Nb14W3O44, Nb16W5O55) using an enumeration-based approach and first-principles density-functional theory calculations. We report common principles governing the cation disorder, lithium insertion mechanism, and electronic structure of these materials. Tungsten preferentially occupies tetrahedral and block-central sites within the block-type crystal structures, and the local structure of the materials depends on the cation configuration. The lithium insertion proceeds via a three-step mechanism, associated with an anisotropic evolution of the host lattice. Our calculations reveal an important connection between long-range and local structural changes: in the second step of the mechanism, the removal of local structural distortions leads to the contraction of the lattice along specific crystallographic directions, buffering the volume expansion of the material. Niobium-tungsten oxide shear structures host small amounts of localized electrons during initial lithium insertion due to the confining effect of the blocks, but quickly become metallic upon further lithiation. We argue that the combination of local, long-range, and electronic structural evolution over the course of lithiation is beneficial to the performance of these materials as battery electrodes. The mechanistic principles we establish arise from the compound-independent crystallographic shear structure and are therefore likely to apply to niobium-titanium oxide or pure niobium oxide crystallographic shear phases.

Long-Range Structural Changes in the Meiotic Nucleus Revealed by Changes in Stress Communication Along the Chromosome

Homologous recombination drives structural reorganization of the nucleus in early meiosis. In order to investigate the connection between homolog pairing, meiotic progression, and the dynamics of the underlying chromatin, we tracked flourescently labeled homolog pairs in synchronized S. cerevisae. Various previously unreported statistics of the anomalous inter-loci motion correlate with meiotic progression and can be quantitatively reproduced by a simple polymer model of the sister chromatids. The first of these is the distribution of waiting times for the homologous loci to come into and out of contact with each other (loosely, inter-locus “looping” and “unlooping” times). The full shape of the looping time distribution can be quantitatively reproduced by a simple model of two polymers diffusing independently in a spherical confinement. This finding suggests a dominant role for diffusion-limited, undirected search in homolog pairing in early meiosis. This is in sharp contrast with the intuition that a heavy-tailed search process could never drive such a critical cell-cycle stage. We further show that the inter-locus velocity-velocity correlation (VVC) quantitatively matches analytical results for the inter-locus VVC of our polymer model, allowing us to leverage our analytical theory to extract the time scale of stress communication between the labeled loci along the chromosome. We show that stresses can take tens of minutes to propagate between loci on paired chromosomes, and that the increasing connectivity between the chromosomes as the cell progresses through meiosis can be quantified by the shortening of this communication time. Our study highlights the power of coarse-grained polymer models to analyze dynamic structural properties of the nucleus in vivo and the importance of analytical theory for uncovering intracellular connections that might be obscured by lag times of many minutes.

Long-Range Conformational Response of a PDZ Domain to Ligand Binding and Release: A Molecular Dynamics Study.

The binding of a ligand to a protein may induce long-range structural or dynamical changes in the biomacromolecule even at sites physically well separated from the binding pocket. A system for which such behavior has been widely discussed is the PDZ2 domain of human tyrosine phosphatase 1E. Here, we present results from equilibrium trajectories of the PDZ2 domain in the free and ligand-bound state, as well as nonequilibrium simulations of the relaxation of PDZ2 after removal of its peptide ligand. The study reveals changes in inter-residue contacts, backbone dihedral angles, and C(α) positions upon ligand release. Our findings show a long-range conformational response of the PDZ2 domain to ligand release in the form of a collective shift of the secondary structure elements α2, β2, β3, α1-β4, and the C terminal loop relative to the rest of the protein away from the N-terminus, and a shift of the loops β2-β3 and β1-β2 in the opposite direction. The shifts lead to conformational changes in the backbone, especially in the β2-β3 loop but also in the β5-α2 and the α2-β6 loop, and are accompanied by changes of inter-residue contacts mainly within the β2-β3 loop as well as between the α2 helix and other segments. The residues showing substantial changes of inter-residue contacts, backbone conformations, or C(α) positions are considered "key residues" for the long-range conformational response of PDZ2. By comparing these residues with various sets of residues highlighted by previous studies of PDZ2, we investigate the statistical correlation of the various approaches. Interestingly, we find a considerable correlation of our findings with several works considering structural changes but no significant correlations with approaches considering energy flow or networks based on inter-residue energies.

Impact of hydrothermal treatment of FEBEX and MX80 bentonites in water, HNO3 and Lu(NO3)3 media: Implications for radioactive waste control

Engineered barriers of deep geological repositories (DGR) are commonly constructed with bentonite. FEBEX and MX80 bentonites have been selected by different countries as reference materials for the sealing of repositories; however, their chemical reactivity with high-level long-lived radioactive wastes (HLRW) under subcritical conditions had not been explored before. The hydrothermal stability in neutral and acid media and chemical reactivity in contact with an actinide analogous compound were both studied. The long-range and short-range structural changes were analyzed by X-ray diffraction, nuclear magnetic resonance and scanning electron microscopy. Both bentonites have exhibited a good stability in neutral and acid media and have generated a new phase immobilizing the actinide analogous compound. The extent of the chemical reaction is higher in MX80 bentonite than in FEBEX bentonite.

Elucidation of the Local and Long-Range Structural Changes that Occur in Germanium Anodes in Lithium-Ion Batteries

Metallic germanium is a promising anode material in secondary lithium-ion batteries (LIBs) due to its high theoretical capacity (1623 mAh/g) and low operating voltage, coupled with the high lithium-ion diffusivity and electronic conductivity of lithiated Ge. Here, the lithiation mechanism of micron-sized Ge anodes has been investigated with X-ray diffraction (XRD), pair distribution function (PDF) analysis, and in-/ex-situ high-resolution 7Li solid-state nuclear magnetic resonance (NMR), utilizing the structural information and spectroscopic fingerprints obtained by characterizing a series of relevant LixGey model compounds. In contrast to previous work, which postulated the formation of Li9Ge4 upon initial lithiation, we show that crystalline Ge first reacts to form a mixture of amorphous and crystalline Li7Ge3 (space group P3212). Although Li7Ge3 was proposed to be stable in a recent theoretical study of the Li–Ge phase diagram (Morris, A. J.; Grey, C. P.; Pickard, C. J. Phys. Rev. B: Condens. Matter Ma...

The linker between the dimerization and catalytic domains of the CheA histidine kinase propagates changes in structure and dynamics that are important for enzymatic activity.

The histidine kinase, CheA, couples environmental stimuli to changes in bacterial swimming behavior, converting a sensory signal to a chemical signal in the cytosol via autophosphorylation. The kinase activity is regulated in the platform of chemotaxis signaling complexes formed by CheW, chemoreceptors, and the regulatory domain of CheA. Our previous computational and mutational studies have revealed that two interdomain linkers play important roles in CheA’s enzymatic activity. Of the two linkers, one that connects the dimerization and ATP binding domains is essential for both basal autophosphorylation and activation of the kinase. However, the mechanistic role of this linker remains unclear, given that it is far from the autophosphorylation reaction center (the ATP binding site). Here we investigate how this interdomain linker is coupled to CheA’s enzymatic activity. Using modern nuclear magnetic resonance (NMR) techniques, we find that by interacting with the catalytic domain, the interdomain linker initiates long-range structural and dynamic changes directed toward the catalytic center of the autophosphorylation reaction. Subsequent biochemical assays define the functional relevance of these NMR-based observations. These findings extend our understanding of the chemotaxis signal transduction pathway.

Hysteretic adsorption of CO₂ onto a Cu₂(pzdc)₂(bpy) porous coordination polymer and concomitant framework distortion.

The present study focuses on the long-range structural changes that occur in the porous coordination polymer Cu2(pzdc)2(bpy) (pzdc = pyrazine-2,3-dicarboxylate, bpy = 4,4'-bipyridine), also known as CPL-2, upon adsorption of CO2 at 25 °C and up to 7 atm. The structural data were gathered using in situ diffraction studies. CPL-2 exhibited an unexpected hysteretic adsorption-desorption process. The onset of hysteresis occurs at a pressure where full occupancy of the volume of the CPL-2 galleries is achieved while the framework retains a structure similar to what is observed under ambient conditions. Moreover, the onset occurs at a CO2 partial pressure larger than 2 atm and could be related to a combination of adsorbate-adsorbent interactions and forces exerted onto the CPL-2 framework. Pore volumes estimated from fits of the Dubinin-Astakhov isotherm model against the CO2 desorption data gathered at 25 and -78.5 °C, respectively, provided further evidence of the aforementioned CPL-2 framework changes. These findings are of relevance to the understanding of adsorption processes in metal organic frameworks or coordination polymers under conditions that are of relevance to gas capture at industrial scale.

Correlative nanoscale imaging of actin filaments and their complexes.

Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

A dynamic model of long‐range conformational adaptations triggered by nucleotide binding in GroEL‐GroES

The molecular chaperone, GroEL, essential for correct protein folding in E. coli, is composed of 14 identical subunits organized in two interacting rings, each providing a folding chamber for non-native substrate proteins. The oligomeric assembly shows positive cooperativity within each ring and negative cooperativity between the rings. Although it is well known that ATP and long-range allosteric interactions drive the functional cycle of GroEL, an atomic resolution view of how ligand binding modulates conformational adaptations over long distances remains a major challenge. Moreover, little is known on the relation between equilibrium dynamics at physiological temperatures and the allosteric transitions in GroEL. Here we present multiple all-atom molecular dynamics simulations of the GroEL-GroES assemblies at different stages of the functional cycle. Combined with an extensive analysis of the complete set of experimentally available structures, principal component analysis and conformer plots, we provide an explicit evaluation of the accessible conformational space of unliganded GroEL. Our results suggest the presence of pre-existing conformers at the equatorial domain level, and a shift of the conformational ensemble upon ATP-binding. At the inter-ring interface the simulations capture a remarkable offset motion of helix D triggered by ATP-binding to the folding active ring. The reorientation of helix D, previously only observed upon GroES association, correlates with a change of the internal dynamics in the opposite ring. This work contributes to the understanding of the molecular mechanisms in GroEL and highlights the ability of all-atom MD simulations to model long-range structural changes and allosteric events in large systems.

The Ryanodine Receptor N-Terminal Disease Hot Spot Intersubunit Interface is Disrupted by Channel Opening and Affected by Disease Mutations Acting via Long-Range Structural Changes

Ryanodine Receptors (RyRs) are intracellular calcium-release channels acting as one of the key regulatory elements in the excitation-contraction coupling. More than 350 mutations have been found in RyRs that are known to underlie severe genetic diseases. Mutations in the skeletal muscle isoform (RyR1) are associated with malignant hyperthermia (MH) and central core disease (CCD), while mutations in the cardiac isoform (RyR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD). Most mutations confer a gain of function, but the precise mechanisms that explain enhanced channel opening up to the molecular scale have remained elusive. Here we present pseudo-atomic models of the N-terminal disease hot spot in the open and closed states of the RyR, along with crystal structures of several disease mutants. The data show that the intersubunit interfaces formed by tetrameric N-terminal disease hot spots are disrupted upon channel opening in wild-type RyRs. This intersubunit interface harbors 19 disease mutations, the largest cluster within the N-terminal region, indicating the vulnerability of this interface in channel regulation. We present crystal structures and thermal stabilities of nine disease mutants located at other interfaces. The effect of most mutations are destabilizing to the protein, with decreases in melting temperatures as large as ∼10°C. Buried disease mutations cause structural changes to the intersubunit interface, while mutations affecting ionic pairing at the intra-subunit interface significantly alter relative domain orientations. Mutations far away from the intersubunit interface can thus affect these contacts via long-range conformational changes. These results illuminate the intersubunit interface between N-terminal disease hot spots as a prime target for disease mutations through direct or indirect conformational changes.

Preparation and solid-state characterization of ball milled saquinavir mesylate for solubility enhancement

Saquinavir is an anti-retroviral drug with very low oral bioavailability (e.g. 0.7–4.0%) due to its affinity toward efflux transporters (P-gp) and metabolic enzymes (CYP3A4). The aim of this study was to characterize the effects of high-energy ball milling on saquinavir solid-state characteristics and aqueous solubility for the design of effective buccal drug delivery systems. The solubility of saquinavir mesylate was evaluated in simulated saliva before and after milling for 1, 3, 15, 30, 50, and 60 h. To elucidate changes in crystallinity and long-range structure in the drug, analyses of the milled powders were performed using XRD, ATR-IR, DSC/TGA, BET surface area, EDX and SEM. In addition, the effects of milling time on saquinavir solubility were statistically correlated using repeated measures ANOVA. Results of this study indicate that the milling of saquinavir mesylate produces nanoporous particles with unique surface structures, thermal properties, and increased aqueous solubility. Optimal milling time occurred at 3 h and corresponded to a 9-fold solubility enhancement in simulated saliva. Thermal analysis revealed only a slight decrease in melting point ( T m) from 242 °C to 236 °C after 60 h milling. XRD diffractograms indicate a gradual crystalline-to-amorphous transition with some residual crystallinity remaining after 60 h milling time. Unstable polymorphic structures appeared between 15 and 30 h which were converted to more stable isomorphs at 60 h. Aggregate formation also seems to occur after 15 h but no metal contamination of the drug was observed during the milling process as determined by EDX analysis. In conclusion, high-energy ball milling may be a method of choice for improving the solubility of saquinavir and facilitating novel drug formulations design.

Low-Temperature FTIR Study of Gloeobacter Rhodopsin: Presence of Strongly Hydrogen-Bonded Water and Long-Range Structural Protein Perturbation upon Retinal Photoisomerization

Gloeobacter rhodopsin (GR) is a light-driven proton-pump protein similar to bacteriorhodopsin (BR), found in Gloeobacter violaceus PCC 7421, a primitive cyanobacterium. In this paper, structural changes of GR following retinal photoisomerization are studied by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy. The initial motivation was to test our hypothesis that proton-pumping rhodopsins possess strongly hydrogen-bonded water molecules in the active center. Water O-D stretching vibrations at <2400 cm(-1) in D(2)O have been regarded as coming from such strongly hydrogen-bonded water, and there is a strong correlation between the proton-pumping activity and the presence of such water molecule. Since GR pumps protons, we expected that GR also possesses strongly hydrogen-bonded water molecule(s), and the FTIR results clearly show that this is indeed the case. In addition, another unexpected finding was gained from the frequency region of protonated carboxylic acids in the GR(K) minus GR spectra at 77 K, where we observed the unique bands of a protonated carboxylic acid at 1735 (+)/1730 (-) cm(-1). Comprehensive mutation study revealed that the vibrational bands originate from the carboxylic C=O stretch of Glu132 at the position corresponding to Asp96 in BR. Glu132 presumably functions as an internal proton donor for the retinal Schiff base, but they may be located far apart (ca. 12 A in BR). The present study demonstrates the long-range structural changes of GR along the proton pathway, even though the protein matrix is frozen at 77 K.

Conformational specificity in allosteric signaling: high‐throughput measurement of modular secondary structural changes within human Eg5 kinesin

The motor domain of human Eg5 kinesin (HsEg5) displays an allosteric pocket at the extracellular surface; binding of monastrol or S‐trityl‐L‐cysteine (STC) to this allosteric site, created by the L5 loop, and subsequent distal conformational changes impairs ATP hydrolysis and cellular function. We examined the roles of E116 and E118 in the L5 loop in allosteric signaling. First, there is a positional correlation on the outcome of amino acid substitutions, irrespective of its nature. Single‐site substitution of E118 increased basal ATP hydrolysis rates of Eg5, whereas substitution of E116 resulted in lower ATPase rates. Thus, sequence variation at residues 116 and 118 of kinesin L5 loop can drive upregulation and downregulation of ATP hydrolysis, respectively. From high‐throughput measurements of the secondary structure composition of proteins in solution, vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Moreover, spectral changes found for E116 variants were similar to those of wildtype Eg5 with monastrol or STC. Therefore, allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady‐state changes to the secondary structure of the Eg5 motor domain in solution, and these rapid methods will provide insight into how long‐range structural changes impact motor function.

Conformational Specificity in Allosteric Signaling: High-throughput Measurement of Modular Secondary Structural Changes within Human Eg5 Kinesin

Communication within allosteric enzymes requires long-range interactions. The ATPase activity of human Eg5 kinesin (HsEg5) is allosterically inhibited upon noncovalent binding of monastrol or S-trityl-L-cysteine (STC) with the L5 loop and subsequent distal conformation changes. Our hypothesis is that the E116 and E118 carboxylates, flanking an isolated β-bridge in the L5 loop, are required for allosteric signaling during small chemical inhibition. Surprisingly, assessment by molecular and biochemical methods revealed that substitution of these carboxylates, irrespective of its nature, has a positional dependence on HsEg5 ATPase rates. Single-site substitution of E118 increased basal ATP hydrolysis rates of Eg5, whereas substitution of E116 resulted in lower ATPase rates. Thus, sequence variation at residues 116 and 118 of the L5 loop can drive upregulation and downregulation of ATP hydrolysis in vitro, respectively. We conducted high-throughput infrared measurements of the secondary structure composition of the upregulated and downregulated proteins in solution. Vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Compared to control samples, in HsEg5 motors with E118 substitutions, amide I′ components between 1642-1620 cm−1 decreased in normalized area, whereas proteins with alterations of E116 exhibited an increased contribution in the amide I′ area for modes between 1684 and 1646 cm−1. Moreover, the spectral changes exhibited by E116 variants were similar to those of wildtype HsEg5 during allosteric inhibition by monastrol or STC. We conclude that allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady-state changes to the secondary structure of the HsEg5 motor domain in solution, and these rapid methods will provide insight into how long-range structural changes impact motor function.

An Empirical Strategy to Detect Spurious Effects in Long Memory and Occasional-Break Processes

Long-range dependence and structural changes in level are intimely related phenomena and it is very difficult to separate the two effects. In this article, we present an empirical procedure to distinguish between long-memory and occasional-break processes. An extensive Monte Carlo experiment illustrates the performance of the procedure and an application to real data is also included.

Structure of the Myristylated Human Immunodeficiency Virus Type 2 Matrix Protein and the Role of Phosphatidylinositol-(4,5)-Bisphosphate in Membrane Targeting

During the late phase of retroviral replication, newly synthesized Gag proteins are targeted to the plasma membrane (PM), where they assemble and bud to form immature virus particles. Membrane targeting by human immunodeficiency virus type 1 (HIV-1) Gag is mediated by the PM marker molecule phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2], which is capable of binding to the matrix (MA) domain of Gag in an extended lipid conformation and of triggering myristate exposure. Here, we show that, as observed previously for HIV-1 MA, the myristyl group of HIV-2 MA is partially sequestered within a narrow hydrophobic tunnel formed by side chains of helices 1, 2, 3, and 5. However, the myristate of HIV-2 MA is more tightly sequestered than that of the HIV-1 protein and does not exhibit concentration-dependent exposure. Soluble PI(4,5)P 2 analogs containing truncated acyl chains bind HIV-2 MA and induce minor long-range structural changes but do not trigger myristate exposure. Despite these differences, the site of HIV-2 assembly in vivo can be manipulated by enzymes that regulate PI(4,5)P 2 localization. Our findings indicate that HIV-1 and HIV-2 are both targeted to the PM for assembly via a PI(4,5)P 2-dependent mechanism, despite differences in the sensitivity of the MA myristyl switch, and suggest a potential mechanism that may contribute to the poor replication kinetics of HIV-2.