1,720,979 research outputs found
Molecular mechanisms of folding of intrinsically disordered proteins
Full text linkIn this thesis, we studied the interaction between the intrinsically disordered domain of the nucleoprotein (N) of Measles virus (MeV), NTAIL, and its partner XD, the X domain of the MeV phosphoprotein (P). It had been previously shown that the α-MoRE (residues 489-506) of NTAIL undergoes an α-helical folding after binding to XD (induced fit mechanism) while regions flanking the α-MoRE remain disordered (fuzzy) in the complex. The fuzzy appendage preceding the α-MoRE was shown to decrease the binding affinities towards XD and the rate of folding of the α-MoRE. In this thesis, by producing NTAIL variants (single-site variants, truncation variants, artificial variants) and performing kinetic experiments of the interaction with XD, we studied the folding after binding mechanism of NTAIL at the single residue level, and investigated the mechanisms through which the fuzzy region hampers the binding affinity and the folding rate of the α-MoRE. We concluded that the central part of the helix is responsible for the initial interactions driving the binding with XD. Moreover, we found that the fuzzy region causes a decrease in the folding rate of the α-MoRE through a combination of entropic and enthalpic effects. We also studied the interaction between NTAIL and a variant of XD, I504A, that populates only the native state. These studies showed that both the binding and the folding steps of the NTAIL-XD interaction are highly dependent on the shape of XD, suggesting that this IDP folds by heterogeneous nucleation via a mechanism induced by the shape of the partner (templated folding)
Rapid kinetics of calcium dissociation from plant calmodulin and calmodulin-like proteins and effect of target peptides
No full textCalcium (Ca2+) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca2+ signals are decoded into defined plant adaptive responses by different Ca2+ sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca2+ decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca2+ dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca2+-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca2+ sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca2+ binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca2+-decoding and Ca2+-controlled target binding mechanisms in plants. (C) 2021 Elsevier Inc. All rights reserved
Mechanism of folding and binding of the n-terminal SH2 domain from SHP2
No full textSHP2 is a phosphatase protein, involved in many cellular pathways, comprising two SH2 domains (namely N-SH2 and C-SH2) and a phosphatase domain. Among others, the interaction between SHP2 and Gab2 (Grb2 associated binder) is critical in cell death and differentiation. SHP2 binds to Gab2 through its SH2 domains, which recognize specific regions of Gab2 characterized by the presence of a phosphorylated tyrosine. In order to shed light on the dynamic and functional properties of this protein-protein interaction, we studied the mechanism of folding of N-SH2 and the binding process to a peptide mimicking a region of Gab2. The data presented represent the first description by stopped-flow of the kinetics of binding of an SH2 domain in solution. By performing experiments at different ionic strengths, we elucidate the electrostatic nature of the interaction, highlighting a key role of the negative charge of the phosphotyrosine in the recognition event of the reaction. Furthermore, by analysing the equilibrium and kinetics of folding of N-SH2 folding we demonstrate the presence of an intermediate along the folding pathway. These results are discussed in the light of previous works on another SH2 domain
Understanding Binding-Induced Folding by Temperature Jump
No full textTemperature jump is a powerful technique for the characterization of fast kinetics and can be readily employed to understand both binding and folding reactions. Here we summarize briefly a temperature-jump prototypical experiment between an intrinsically disordered protein and its physiological partner. The model used is the NTAIL domain from Measles virus Nucleoprotein and its natural ligand, the globular PXD domain from Measles virus Phosphoprotein. We recapitulate how to set up the experiment and how to analyze data in order to extract the kinetic parameters of the reaction
Folding and misfolding of a PDZ tandem repeat
No full textAlthough the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of whirlin (PDZ1 and PDZ2 - namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics
Probing the effects of local frustration in the folding of a multidomain protein
No full textOur current knowledge of protein folding is primarily based on experimental data obtained from isolated domains. In fact, because of their complexity, multidomain proteins have been elusive to the experimental characterization. Thus, the folding of a domain in isolation is generally assumed to resemble what should be observed for more complex structural architectures. Here we compared the folding mechanism of a protein domain in isolation and in the context of its supramodular multidomain structure. By carrying out an extensive mutational analysis we illustrate that while the early events of folding are malleable and influenced by the absence/presence of the neighboring structures, the late events appear to be more robust. These effects may be explained by analyzing the local frustration patterns of the domain, providing critical support for the funneled energy landscape theory of protein folding, and highlighting the role of protein frustration in sculpting the early events of the reaction
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Templated folding of intrinsically disordered proteins
No full textMuch of our current knowledge of
biological chemistry is founded in the
structure-function relationship, whereby
sequence determines structure that
determines function. Thus, the discovery
that a large fraction of the proteome is
intrinsically disordered, while being
functional,
has
revolutionized
our
understanding of proteins and raised new
and
interesting
questions.
Many
intrinsically disordered proteins (IDPs)
have been determined to undergo a
disorder to order transition when
recognizing their physiological partners,
suggesting their mechanisms of folding are
intrinsically different from those observed
in globular proteins. However, IDPs also
follow some of the classic paradigms
established for globular proteins, pointing
to important similarities in their behavior.
In this review, we compare and contrast the
folding mechanisms of globular proteins
with the emerging features of binding-
induced folding of intrinsically disordered
order transitions of intrinsically disordered
proteins appear to follow rules of globular
protein folding such as the cooperative
nature of the reaction, their folding
pathways are remarkably more malleable,
due to the heterogeneous nature of their
folding nuclei, as probed by analysis of
linear free energy relationship plots. These
insights have led to a new model for the
disorder-to-order transition in IDPs termed
‘templated folding’, whereby the binding
partner dictates distinct structural
transitions en route to product, while
ensuring a co-operative folding
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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