“Intrinsic disorder” is generally referred to the conformational status of native proteins lacking of secondary and/or tertiary structure, although not exposed to any denaturing agent. These proteins, which are called intrinsically disordered (IDP/IDRs) represent a large class in the proteomes of all living beings, with a remarkable abundance among more complex eukaryotes and viruses. IDPs have been recognized to be involved in many relevant physiological and pathological functions, such as the coacervation of membrane-less organelles or the fibrillation in amyloid bodies. It is becoming clearer that fast and massive intermolecular interactions involving IDPs are governing both kinds of phenomena and that pathologies can arise from dysregulations of conformational properties and aggregation ability. The conformation and aggregation features of IDPs have been ascribed in turn to several factors, such as sequence length, hydrophobic interactions, hydrogen bonds or electrostatic charges. The latter deserve particular attention since charged residues are particularly abundant in IDPs. The net charge per residue (NCPR), the total fraction of charged residues (FCR), and the linear distribution of opposite charges (κ value) have been recently regarded as the primary determinants of IDPs conformational properties. The first part of the experimental work presented in this thesis was inspired by the concept of NCPR, which represents the net charge normalized by the protein length. The aim is to describe how the NCPR influences the ability of IDPs to respond to environment pH changes through loss of solubility. PNT from measles virus was used as a model IDP. Moreover, the wild type (wt) protein was compared with an array of PNT variants sharing the same hydrophobicity and total number of charged residues (FCR), but differing in net charges per residue and isoelectric points (pI). Tested proteins showed a solubility minimum close to their pI, as expected, but the pH-dependent decrease of solubility was not uniform and driven by the NCPR of each variant. Our data suggest that the overall solubility of a protein can be dictated by protein regions endowed with NCPR and, hence, prompter to respond to pH changes. The second part of experimental work was inspired by the concept of charge clustering. The aim was consisting at verifying that the compaction properties of IDPs are tunable by the κ value. We have used two well-characterized IDPs, namely measles virus NTAIL and Hendra virus PNT4, as model systems. Taking advantage of the high sequence designability of IDPs, genes of PNT4 and NTAIL were redesigned to obtain two sets of synthetic proteins each including the wild type (wt) form and two “κ variants”. In low-κ variants, charged amino acids are most evenly distributed, in high-κ variants charges are clustered as much as possible at the N- and C-termini (high κ). κ variants, along with wt forms, were subjected to various biophysical techniques to assess their conformational properties.Overall, experimental data confirm the expected trend, with compactness increasing with κ value. The increase of compactness does not follow a general trend, but it is protein-specific and related to the proline content. All together, these findings confirm previous theoretical and experimental data on the role of charged residues frequency (NCPR) and distribution (κ). The main value of this experimental work is in pinpointing the context, which is the environment – pH – or the amino acid composition – proline % –, where such driving forces of aggregation and compaction are mostly effective. This knowledge is useful not only to describe how the conformational behavior of IDPs is encoded by their amino acid sequence, but also to rationally design non-natural IDPs with desired conformational and aggregation properties

Intrinsecamente disordinata” viene definita una proteina nativa priva di struttura secondaria o terziaria, non esposta ad agenti denaturanti. Le proteine con queste caratteristiche sono indicate come IDP/IDR, e rappresentano una ampia porzione del proteoma di tutti gli esseri viventi. Le IDP sono coinvolte in molte funzioni fisiologiche e patologiche, come la formazione di organuli cellulari privi di membrane e nei processi di fibrillazione associate ad amiloidosi. Entrambi questi fenomeni appaiono sempre più associati alla capacità delle IDP di formare interazioni intermolecolari. Stati patologici possono essere causati da disfunzioni e cattiva regolazione delle proprietà conformazionali e di aggregazione delle IDP. L’aggregazione e la conformazione delle IDP sono state ascritte a diversi fattori: la lunghezza della sequenza, le interazioni idrofobiche, i legami ad idrogeno e le cariche elettrostatiche. A questa ultima abbiamo rivolto la nostra attenzione dal momento che le IDP sono ricche di amminoacidi carichi. La carica netta per residuo (NCPR), la frazione totale di residui carichi (FCR) e la distribuzione di residui di carica opposta (valore κ) sono stati considerati i principali determinanti della dimensione della catena e delle classi conformazionali delle IDP. La prima parte del piano sperimentale interessa il concetto di NCPR, cioè la carica netta normalizzata per la lunghezza della proteina. Il nostro obiettivo è di descrivere come questo parametro influenzi la risposta delle IDP a cambiamenti di pH, con conseguente perdita di solubilità. Come modello viene utilizzata PNT del virus del morbillo ed a partire da questa si ottiene un array di varianti aventi la stessa idrofobicità e FCR, ma differenti per NCPR ed il punto isoelettrico (pI). Le proteine analizzate mostrano solubilità minima in corrispondenza del loro valore di pI, come atteso, ma tale diminuzione di solubilità non è uniforme, ma guidata dal valore di NCPR di ciascuna variante proteica. I nostri dati suggeriscono che la solubilità complessiva della proteina sia legata al valore di NCPR. La seconda parte del lavoro si è ispirata al concetto di clusterizzazione di cariche ed ha come obiettivo la valutazione di come le proprietà di compattezza delle IDP dipendano dal valore di κ. In questo caso sono state utilizzate due IDP ben caratterizzate, NTAIL dal virus del morbillo e PNT4 da Hendra virus. Grazie alla possibilità di modificare la sequenza amminoacidica delle IDP, senza interferire sul complessivo disordine strutturale, entrambi i geni sono stati riprogettati. Sono stati ottenuti due set di proteine sintetiche, ciascuno contenente una proteina wt e due varianti in cui le cariche sono uniformemente distribuite (low κ) o completamente segregate all’N- ed al C-terminus (high κ). Le proprietà conformazionali della proteina wt e delle corrispondenti varianti sono valutate mediante tecniche biofisiche. Complessivamente i dati sperimentali confermano il trend atteso cioè un aumento del grado di compattezza conformazionale all’aumentare dei valori di κ, secondo una proporzione che è tipica di ciascuna proteina in relazione al suo contenuto di proline. Complessivamente i nostri risultati confermano precedenti dati computazionali e sperimentali suggerendo come residui carichi, attraverso la loro frequenza (NCPR) e la distribuzione (κ) influenzino solubilità e compattezza delle IDP. I due lavori sperimentali sottolineano il contesto, ambientale (ad esempio le condizioni di pH) o di sequenza (la % di proline), in cui NCPR e distribuzione di cariche sono più efficaci nel determinare le caratteristiche di solubilità e compattezza conformazionale delle IDP. La rilevanza di queste informazioni è legata non solo allo studio IDP naturali, ma anche alla progettazione razionale di proteine non naturali con proprietà aggregative e conformazionali ben definite

(2018). Effect of electrostatic charges on aggregation and conformation of intrinsically disordered proteins . (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).

Effect of electrostatic charges on aggregation and conformation of intrinsically disordered proteins 

TEDESCHI, GIULIA
2018

Abstract

“Intrinsic disorder” is generally referred to the conformational status of native proteins lacking of secondary and/or tertiary structure, although not exposed to any denaturing agent. These proteins, which are called intrinsically disordered (IDP/IDRs) represent a large class in the proteomes of all living beings, with a remarkable abundance among more complex eukaryotes and viruses. IDPs have been recognized to be involved in many relevant physiological and pathological functions, such as the coacervation of membrane-less organelles or the fibrillation in amyloid bodies. It is becoming clearer that fast and massive intermolecular interactions involving IDPs are governing both kinds of phenomena and that pathologies can arise from dysregulations of conformational properties and aggregation ability. The conformation and aggregation features of IDPs have been ascribed in turn to several factors, such as sequence length, hydrophobic interactions, hydrogen bonds or electrostatic charges. The latter deserve particular attention since charged residues are particularly abundant in IDPs. The net charge per residue (NCPR), the total fraction of charged residues (FCR), and the linear distribution of opposite charges (κ value) have been recently regarded as the primary determinants of IDPs conformational properties. The first part of the experimental work presented in this thesis was inspired by the concept of NCPR, which represents the net charge normalized by the protein length. The aim is to describe how the NCPR influences the ability of IDPs to respond to environment pH changes through loss of solubility. PNT from measles virus was used as a model IDP. Moreover, the wild type (wt) protein was compared with an array of PNT variants sharing the same hydrophobicity and total number of charged residues (FCR), but differing in net charges per residue and isoelectric points (pI). Tested proteins showed a solubility minimum close to their pI, as expected, but the pH-dependent decrease of solubility was not uniform and driven by the NCPR of each variant. Our data suggest that the overall solubility of a protein can be dictated by protein regions endowed with NCPR and, hence, prompter to respond to pH changes. The second part of experimental work was inspired by the concept of charge clustering. The aim was consisting at verifying that the compaction properties of IDPs are tunable by the κ value. We have used two well-characterized IDPs, namely measles virus NTAIL and Hendra virus PNT4, as model systems. Taking advantage of the high sequence designability of IDPs, genes of PNT4 and NTAIL were redesigned to obtain two sets of synthetic proteins each including the wild type (wt) form and two “κ variants”. In low-κ variants, charged amino acids are most evenly distributed, in high-κ variants charges are clustered as much as possible at the N- and C-termini (high κ). κ variants, along with wt forms, were subjected to various biophysical techniques to assess their conformational properties.Overall, experimental data confirm the expected trend, with compactness increasing with κ value. The increase of compactness does not follow a general trend, but it is protein-specific and related to the proline content. All together, these findings confirm previous theoretical and experimental data on the role of charged residues frequency (NCPR) and distribution (κ). The main value of this experimental work is in pinpointing the context, which is the environment – pH – or the amino acid composition – proline % –, where such driving forces of aggregation and compaction are mostly effective. This knowledge is useful not only to describe how the conformational behavior of IDPs is encoded by their amino acid sequence, but also to rationally design non-natural IDPs with desired conformational and aggregation properties
BROCCA, STEFANIA
IDP;; conformation;; aggregation;; charged-residues;; proline
IDP;; conformation;; aggregation;; charged-residues;; proline
BIO/10 - BIOCHIMICA
English
1-mar-2018
BIOLOGIA E BIOTECNOLOGIE - 93R
30
2016/2017
open
(2018). Effect of electrostatic charges on aggregation and conformation of intrinsically disordered proteins . (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/198946
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