Gianluca Lattanzi

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

We use single-particle tracking to study the elastic properties of single microtubules grafted to a substrate. Thermal fluctuations of the free microtubule’s end are recorded, in order to measure position distribution functions from which we calculate the persistence length of microtubules with contour lengths between 2.6 and 48 μm. We find the persistence length to vary by more than a factor of 20 over the total range of contour lengths. Our results support the hypothesis that shearing between protofilaments contributes significantly to the mechanics of microtubules. PACS numbers: 87.15.Ya, 87.15.La, 87.16.Ka, 36.20.Ey ∗ Corresponding author. FP and GL have equally contributed to this work.

Capacitance-modulated transistor detects odorant binding protein chiral interactions

Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand–protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein–ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

Conformations of Confined Biopolymers

Nanoscale and microscale confinement of biopolymers naturally occurs in cells and has been recently achieved in artificial structures designed for nanotechnological applications. Here, we present an extensive theoretical investigation of the conformations and shape of a biopolymer with varying stiffness confined to a narrow channel. Combining scaling arguments, analytical calculations, and Monte Carlo simulations, we identify various scaling regimes where master curves quantify the functional dependence of the polymer conformations on the chain stiffness and strength of confinement.

Mechanism of Action of Cyclophilin A Explored by Metadynamics Simulations

Trans/cis prolyl isomerisation is involved in several biological processes, including the development of numerous diseases. In the HIV-1 capsid protein (CA), such a process takes place in the uncoating and recruitment of the virion and is catalyzed by cyclophilin A (CypA). Here, we use metadynamics simulations to investigate the isomerization of CAs model substrate HAGPIA in water and in its target protein CypA. Our results allow us to propose a novel mechanistic hypothesis, which is finally consistent with all of the available molecular biology data.

A new gating site in human aquaporin-4: Insights from molecular dynamics simulations.

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373-385], our analysis on 200ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.

Three-Dimensional Microtubule Behavior in Xenopus Egg Extracts Reveals Four Dynamic States and State-Dependent Elastic Properties

Although microtubules are key players in many cellular processes, very little is known about their dynamic and mechanical properties in physiological three-dimensional environments. The conventional model of microtubule dynamic instability postulates two dynamic microtubule states, growth and shrinkage. However, several studies have indicated that such a model does not provide a comprehensive quantitative and qualitative description of microtubule behavior. Using three-dimensional laser light-sheet fluorescence microscopy and a three-dimensional sample preparation in spacious Teflon cylinders, we measured microtubule dynamic instability and elasticity in interphase Xenopus laevis egg extracts. Our data are inconsistent with a two-state model of microtubule dynamic instability and favor an extended four-state model with two independent metastable pause states over a three-state model with a single pause state. Moreover, our data on kinetic state transitions rule out a simple GTP cap model as the driving force of microtubule stabilization in egg extracts on timescales of a few seconds or longer. We determined the three-dimensional elastic properties of microtubules as a function of both the contour length and the dynamic state. Our results indicate that pausing microtubules are less flexible than growing microtubules and suggest a growth-speed-dependent persistence length. These data might hint toward mechanisms that enable microtubules to efficiently perform multiple different tasks in the cell and suggest the development of a unified model of microtubule dynamics and microtubule mechanics.

Challenging AQP4 druggability for NMO-IgG antibody binding using molecular dynamics and molecular interaction fields.

Neuromyelitis optica (NMO) is a multiple sclerosis-like immunopathology disease affecting optic nerves and the spinal cord. Its pathological hallmark is the deposition of a typical immunoglobulin, called NMO-IgG, against the water channel Aquaporin-4 (AQP4). Preventing NMO-IgG binding would represent a valuable molecular strategy for a focused NMO therapy. The recent observation that aspartate in position 69 (D69) is determinant for the formation of NMO-IgG epitopes prompted us to carry out intensive Molecular Dynamics (MD) studies on a number of single-point AQP4 mutants. Here, we report a domino effect originating from the point mutation at position 69: we find that the side chain of T62 is reoriented far from its expected position leaning on the lumen of the pore. More importantly, the strength of the H-bond interaction between L53 and T56, at the basis of the loop A, is substantially weakened. These events represent important pieces of a clear-cut mechanistic rationale behind the failure of the NMO-IgG binding, while the water channel function as well as the propensity to aggregate into OAPs remains unaltered. The molecular interaction fields (MIF)-based analysis of cavities complemented MD findings indicating a putative binding site comprising the same residues determining epitope reorganization. In this respect, docking studies unveiled an intriguing perspective to address the future design of small drug-like compounds against NMO. In agreement with recent experimental observations, the present study is the first computational attempt to elucidate NMO-IgG binding at the molecular level, as well as a first effort toward a less elusive AQP4 druggability.

Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica autoantibodies.

Background: Neuromyelitis optica autoantibodies target the aquaporin-4 (AQP4) aggregate named orthogonal arrays of particles (OAP). Results: Mutation of AQP4 aspartate 69 (Asp69) impairs NMO-IgG binding leaving the water channel function unaltered as well as its aggregation into OAPs. Conclusion: Asp69 is the key determinant for the formation of NMO-IgG epitopes. Significance: Such evidence provides additional clues on NMO pathogenesis. Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp69) of TM2 for NMO-IgG epitope assembly. Mutation of Asp69 to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp69 mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D69H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp69 is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.

G protein inactive and active forms investigated by simulation methods

Molecular dynamics and computational alanine scanning techniques have been used to investigate G proteins in their inactive state (the Gαi1β1γ2 heterotrimer) as well as in their empty and monomeric active states (Gαi1 subunit). We find that: (i) the residue Q204 of Gαi1 plays a key role for binding Gβ1γ2 and is classified among the most relevant in the interaction with a key cellular partner, the so‐called regulator of G protein signaling protein. The mutation of this residue to L, which is observed in a variety of diseases, provides still fair stability to the inactive state because of the formation of van der Waals interactions. (ii) The empty state turns out to adopt some structural features of the active one, including a previously unrecognized rearrangement of a key residue (K46). (iii) The so‐called Switch IV region increases its mobility on passing from the empty to the active state, and, even more, to the inactive state. Such change in mobility could be important for its several structural and functional roles. (iv) A large scale motion of the helical domain in the inactive state might be important for GDP release upon activation by GPCR, consistently with experimental data. Proteins 2009.

Master equation approach to molecular motors.

A master equation approach to molecular motors allows us to describe a mechanochemical cyclic system where chemical and translational degrees of freedom are treated on an equal footing. A generalized detailed balance condition in the out-of-equilibrium regime is shown to be compatible with the Fokker-Planck equation in the continuum limit. The Onsager reciprocity relations hold for stationary states close to equilibrium, provided the generalized detailed balance condition is satisfied. Semiphenomenological considerations in the case of motor proteins lead to a discrete kinetics model, for which interesting observable quantities may be directly calculated and compared with experimental data.