108 research outputs found
Computational approaches to shed light on molecular mechanisms in biological processes
Computational approaches based on Molecular Dynamics simulations, Quantum Mechanical methods and 3D Quantitative Structure-Activity Relationships were employed by computational chemistry groups at the University of Milano-Bicocca to study biological processes at the molecular level. The paper reports the methodologies adopted and the results obtained on Aryl hydrocarbon Receptor and homologous PAS proteins mechanisms, the properties of prion protein peptides, the reaction pathway of hydrogenase and peroxidase enzymes and the defibrillogenic activity of tetracyclines. © Springer-Verlag 2007
Cholesterol impairment contributes to neuroserpin aggregation
Intraneural accumulation of misfolded proteins is a common feature of several
neurodegenerative pathologies including Alzheimer's and Parkinson's diseases,
and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). FENIB is
a rare disease due to a point mutation in neuroserpin which accelerates protein
aggregation in the endoplasmic reticulum (ER). Here we show that cholesterol
depletion induced either by prolonged exposure to statins or by inhibiting the
sterol regulatory binding-element protein (SREBP) pathway also enhances
aggregation of neuroserpin proteins. These findings can be explained
considering a computational model of protein aggregation under non-equilibrium
conditions, where a decrease in the rate of protein clearance improves
aggregation. Decreasing cholesterol in cell membranes affects their biophysical
properties, including their ability to form the vesicles needed for protein
clearance, as we illustrate by a simple mathematical model. Taken together,
these results suggest that cholesterol reduction induces neuroserpin
aggregation, even in absence of specific neuroserpin mutations. The new
mechanism we uncover could be relevant also for other neurodegenerative
diseases associated with protein aggregation.Comment: 7 figure
The development of biomolecular Raman optical activity spectroscopy
Following its first observation over 40 years ago, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light or, equivalently, the intensity of a small circularly polarized component in the scattered light using incident light of fixed polarization, has evolved into a powerful chiroptical spectroscopy for studying a large range of biomolecules in aqueous solution. The long and tortuous path leading to the first observations of ROA in biomolecules in 1989, in which the author was closely involved from the very beginning, is documented, followed by a survey of subsequent developments and applications up to the present day. Among other things, ROA provides information about motif and fold, as well as secondary structure, of proteins; solution structure of carbohydrates; polypeptide and carbohydrate structure of intact glycoproteins; new insight into structural elements present in unfolded protein sequences; and protein and nucleic acid structure of intact viruses. Quantum chemical simulations of observed Raman optical activity spectra provide the complete three-dimensional structure, together with information about conformational dynamics, of smaller biomolecules. Biomolecular ROA measurements are now routine thanks to a commercial instrument based on a novel design becoming available in 2004
Molecular mechanisms of amyloid self-regulation
Amyloid is associated with both pathological protein deposits and the formation of
functional protein structures. Therefore, several strategies have evolved to control the
formation or inhibition of amyloid in vivo. In this thesis, three separate systems were
investigated in which amyloidogenic protein segments are coupled to regulatory
elements that prevent or promote fibrillation. We describe the molecular mechanism for
how (a) a propeptide segment prevents the uncontrolled aggregation of the mature
peptide, (b) a chaperone domain inhibits amyloid formation, and (c) a pH-dependent
relay controls protein assembly. For this purpose, mass spectrometry (MS)-based
approaches to structural biology were applied and extended, involving gas phase
interaction studies and hydrogen/deuterium exchange MS.
(a) Proinsulin C-peptide is beneficial for the preservation of insulin activity. We show
that C-peptide interferes with insulin amyloid fibril formation at low pH and how
conserved glutamate residues in C-peptide mediate reversible co-precipitation with
insulin. A mechanism is proposed for how the balance between zinc and C-peptide
mediates sorting of insulin into slow acting and rapid acting forms inside the secretory
granules of the pancreatic -cells, which potentially links C-peptide to diabetes type 1
and 2.
(b) Lung surfactant protein C (SP-C) is a highly amyloidogenic transmembrane
polypeptide that controls surface tension in the alveolar phospholipid bilayer. Its
proprotein includes a conserved chaperone domain termed BRICHOS, which is also
associated with neurodegenerative disorders. It is shown here how BRICHOS and its
N-terminal linker recognize hydrophobic residues and trap the SP-C segment in a -
hairpin conformation to prevent amyloid formation.
(c) Spider silk is synthesized as a highly soluble protein that assembles into silk in a
pH-dependent fashion. It is shown that the spider silk protein N-terminal (NT) domain
dimerizes at the same pH interval that triggers silk assembly, and we define the
associated structural changes. Furthermore, the use of the NT domain as a solubility
tag for the expression of aggregation-prone proteins is demonstrated.
In summary, we have determined the molecular basis for three distinct mechanisms by
which fibril formation is controlled through autoregulatory elements and provide insights
into nature’s strategies to control amyloid formation and prevention. Based on these
findings, we can now make conclusions about nature’s handling of amyloidogenic
proteins and their function in general
Rationalisation of the Differences between APOBEC3G Structures from Crystallography and NMR Studies by Molecular Dynamics Simulations
The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal β-strand, β2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the β2 strand for those structures that have a distorted starting conformation of β2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the β2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA
Metal ions and protein folding: conformational and functional interplay
Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia QuÃmica e Biológica, Universidade Nova de LisboaMetal ions are cofactors in about 30% of all proteins, where they fulfill
catalytical and structural roles. Due to their unique chemistry and
coordination properties they effectively expand the intrinsic polypeptide
properties (by participating in catalysis or electron transfer reactions),
stabilize protein conformations (like in zinc fingers) and mediate signal
transduction (by promoting functionally relevant protein conformational
changes). However, metal ions can also exert have deleterious effects in
living systems by incorporating in non-native binding sites, promoting
aberrant protein aggregation or mediating redox cycling with generation of
reactive oxygen and nitrogen species. For this reason, the characterization of
the roles of metal ions as modulators of protein conformation and stability
provides fundamental knowledge on protein folding properties and is
instrumental in establishing the molecular basis of disease. In this thesis we
have analyzed protein folding processes using model protein systems
incorporating covalently bound metal cofactors – iron-sulfur (FeS) proteins –
or where metal ion binding is reversible and associated conformational
readjustments – the S100 proteins.(...
Complexity and Specificity of Sec61-Channelopathies: Human Diseases Affecting Gating of the Sec61 Complex
The rough endoplasmic reticulum (ER) of nucleated human cells has crucial functions in
protein biogenesis, calcium (Ca2+) homeostasis, and signal transduction. Among the roughly one
hundred components, which are involved in protein import and protein folding or assembly, two
components stand out: The Sec61 complex and BiP. The Sec61 complex in the ER membrane represents
the major entry point for precursor polypeptides into the membrane or lumen of the ER and provides
a conduit for Ca2+ ions from the ER lumen to the cytosol. The second component, the Hsp70-type
molecular chaperone immunoglobulin heavy chain binding protein, short BiP, plays central roles
in protein folding and assembly (hence its name), protein import, cellular Ca2+ homeostasis, and
various intracellular signal transduction pathways. For the purpose of this review, we focus on
these two components, their relevant allosteric effectors and on the question of how their respective
functional cycles are linked in order to reconcile the apparently contradictory features of the ER
membrane, selective permeability for precursor polypeptides, and impermeability for Ca2+. The
key issues are that the Sec61 complex exists in two conformations: An open and a closed state
that are in a dynamic equilibrium with each other, and that BiP contributes to its gating in both
directions in cooperation with different co-chaperones. While the open Sec61 complex forms an
aqueous polypeptide-conducting- and transiently Ca2+-permeable channel, the closed complex
is impermeable even to Ca2+. Therefore, we discuss the human hereditary and tumor diseases
that are linked to Sec61 channel gating, termed Sec61-channelopathies, as disturbances of selective
polypeptide-impermeability and/or aberrant Ca2+-permeability
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