Fluorescent Probes and Protein Misfolding: Methods and Applications

Abstract

Protein misfolding is a frequently occurring event in living cells and without the quality control, the consequences are disasterous. In this thesis, we studied protein misfolding in vitro by employing a variety of fluorophores, to either detect the highly ordered aggregates of intrinsically disordered proteins/peptides or slight misfolding of a natively folded protein.

Misfolding and concurrent self-assembly of amyloid-beta (Aβ) peptide is believed to be the cause of Alzheimer’s disease. Kinetics of fibril formation correlates with the aggressiveness of a particular variant of Aβ peptide. Hence following the fibril formation in vitro provides a valuable information about the intrinsic and extrinsic factors that play role in the disease development. The most common way of following the amyloid fibril formation in vitro is ThT assay. ThT is a non-covalent fluorescent probe that mainly has affinity for β-sheets. However, unspecific ThT binding to small molecules poses a problem. Here, we developed an alternative assay for ThT, where the mixed aggregates of fluorophore-labeled and unlabeled Aβ are discriminated by their size using a filter-trap and subsequently detected. Signature amyloid fibril formation curves were successfully obtained using filter-trap method, containing information about different stages of fibril formation. We benchmarked the method with two ThT-interfering substances, human serum albumin and negatively charged nanoparticles.

α-synuclein is also an intrinsically disordered protein, playing a central role in Parkinson’s disease pathology. Here, we followed the fibril formation kinetics with ANS, which has affinity to the hydrophobic surfaces on protein, in addition to ThT. ANS reproduces the signature sigmoidal curve, even though it does not have affinity to β-sheets, but hydrophobic patches. This is an implication pointing out that stacking β-sheets and exposure of the hydrophobic patches happen simultaneously. In addition, we investigated the effect of negatively charged vesicles and found that they accelerate the aggregate formation and the surface properties of the aggregates in ratio (lipid/protein) dependent manner. Moreover, lipids were found to be taken up during α-synuclein aggregation up to a certain L/P ca.14.

Valuable information about the hot-spots in the amyloid peptide sequence is obtained via model- and disease related mutations. In second paper, we designed two single mutations for Aβ40 and Aβ42 that contained substitutions from lysine to arginine. We assessed the kinetic effect, microscopic rate constants, structural compatibility by coaggregation and cross-seeding (with wild-type) of these mutated variants as well as the morphology of formed fibrils. We found that Lys-Arg substitution retards the fibril formation in both positions (K16R and K28R). K16R mutation was more effective than K28R, however both mutants shared a common selective hinderance mechanism; a much slower elongation rate. We also found that the wild-type and the mutants are fully compatible with each other in coaggregation and cross-seeding studies and the fibril morphology changes for K28R mutation.

Nanoparticles also induce protein misfolding. In the first study of the second part of the thesis, we developed a method that points the interacting nanoparticle-protein partners. The method employs solvatochromic dyes, as they have affinity to hydrophobic patches, therefore the changes in the intensity with respect to the controls can be correlated with protein misfolding. The method is fully capable of reporting the changes over a time period from miliseconds to days. Moreover, the colloidal stability may also be monitored.

Finally, we invesitaged the protein adsorption to nanoparticles in-depth by partly using the screening method developed. Here, we brought together the most important concepts that play role in protein adsorption (i.e., protein stability, nanoparticle size and surface chemistry) and developed a simple model for the adsorption and misfolding kinetics of proteins on the surface with an emphasis on the dynamics of protein misfolding at different surfaces.

Abstract (Swedish)

Popular Abstract in English

Proteins are smart molecules. They act in a vast variety of roles, for instance as transporters, messengers, catalysts, warriors and scaffolds. The ability of a specific protein to perform a task is closely related to its three-dimensional shape (native fold). However, not all proteins encoded in human genome have a structure; some of them are devoid of a well-defined structure -kind of like a loose threads. These proteins are called intrinsically disordered proteins (IDP). Under certain conditions, both natively folded and intrinsically disordered proteins can "misfold". Protein misfolding is a forbidden path with very serious consequences to vitality. Despite of the risk that misfolding bears, proteins can misfold at any instance in the body and we are rescued by our own body’s quality control system.

This thesis is based on the in test tube (in vitro) detection of protein misfolding, induced either intrinsically or extrinsically. The detection is achieved by small reporter molecules, either linked to the protein covalently or bound non-covalently.



Misfolding of some class of IDPs cause neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Under certain conditions, they can misfold and assemble into highly-ordered structures with a large aspect ratio, which are called fibrils. Small soluble species formed on pathway of those fibrils or the self-assembly process are now the two accepted pathways that lead to the toxicity. Understanding the fibril formation process in vitro, broadens our horizon regarding the possible ways of combating the disease.



The conventional way of studying the amyloid fibril formation in vitro includes a non-covalent fluorescent dye ThT, that has affinity to the smallest characteristic structural unit of a fibril. In the first paper, we developed an alternative in vitro method for the detection of amyloid fibrils formation. The method aids for the situations where ThT detection is not applicable; such as when investigating the effect of small molecules (that unspecifically bind ThT) on the fibril formation kinetics. Our approach was to discriminate the aggregates according to their size with a filter membrane. Detection was achieved by tagging the amyloid peptide covalently with a fluorophore. We succeeded to reproduce the signature ThT signal for amyloid fibril formation kinetics, that can report on various stages of this kinetic process.



Amyloid peptide/protein's sequence encodes how rapidly it forms fibrils. Studying the sequence determinants of this process also contributes to the understanding of the disease. In the second paper, we designed model mutations within the amyloid-beta sequence that allow us to analyze the importance of those residues. These single mutations were conservative, meaning that the substituted amino acid has similar physical properties to the original one. We found that such conservative mutation hinders greatly the formation of fibrils by selectively slowing down the rates of elongation. Aside from the relative importance of the amino acids in the sequence, targeting such residues may be a way to combat the disease.



The third project deals with the Parkinson's disease protein alpha-synuclein and its interaction with lipid vesicles. Here, we additionally monitored the amyloid fibril formation process with another non-covalent dye, ANS, that has affinity to hydrophobic patches. We incorporated the novel way of detection in monitoring the alpha-syn fibril formation in presence of lipid vesicles. We found that ANS monitors the process as accurate as ThT. Furthermore, the presence of negatively charged vesicles accelerated the amyloid fibril formation. In the end of the experiments, we also found that the lipids were taken up by fibrils, therefore vesicles were not intact anymore. Overall, our results imply that the surface and kinetic properties of alpha-syn aggregates/aggregation process is altered by the presence of the vesicles. Our results further verify that the relevance and the importance of the negatively charged lipids on the pathology of the disease.



Furthermore, we continued the detection of misfolding with ANS in a completely different system. This time, the ability of ANS and Nile Red (another probe that is specific for hydrophobic patches) to detect misfolding was used for screening nanoparticle induced misfolding of proteins. Nanoparticles come in various geometries and their dimensions are in the scale of one hundredth of the thickness of human hair. Industrialization brought nanoparticles to our lives and every day we take up nanoparticles voluntarily or involuntarily. Thereby, they can be harmful. Indeed, most proteins bind to nanoparticles and experience binding-induced misfolding. The method developed in the fourth paper aimed to find the nanoparticle-protein pairs that interact. Additionally, the changes in the protein intactness can be followed over a time period. The simple and adaptable nature of the method makes it ideal for its employment for initial screens in nanoparticle-based biological applications.



Finally, we studied the interactions of a positive-hit nanoparticle-protein pair that we found in the benchmark of fourth paper. The stability of a protein determines how vulnerable the native-fold that protein adopts is. Here, we used two variants of an enzyme, human carbonic anhydrase, differing in stability. Human carbonic anhydrase converts the carbon dioxide to bicarbonate and not coincidentally is one of the fastest enzyme in human body. We found that these enzymes misfold and lose their function more rapidly in presence of negatively charged plastic nanoparticles (water-hating) as compared to negatively charged silica nanoparticles (water-loving), regardless of their stability. This is related to the barriers that proteins need to overcome in order to be on the surface and adapt its structure. Water-loving or -hating features of the nanoparticle surface also define the non-native paths that protein can adopt; i.e. water-hating surfaces impose slight structural rearrangements while water-loving surfaces trigger large structural-rearrangements.

• author: Nasir, Irem
• supervisor: Celia Cabaleiro-Lago
• opponent: Assist. Prof. Esbjörner-Winters, Elin, Chalmers Institute of Technology
• organization: Biochemistry and Structural Biology; NanoLund: Centre for Nanoscience
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Biochemistry and Molecular Biology
• keywords: Protein misfolding, fluorophores, filter-trap, site-specific fluorophore labeling, amyloid-beta, microscopic rate constants, solvatochromic probes, alpha-synuclein, lipid vesicles, nanoparticles, protein adsorption
• pages: 166 pages
• defense location: KC K:B
• defense date: 2015-12-04 09:15:00
• ISBN: 978-91-7422-416-0
• language: English
• LU publication?: yes
• id: 1897a9a6-92b3-4eb3-b6ee-0301cb6ba731 (old id 8168647)
• date added to LUP: 2016-04-04 10:19:39
• date last changed: 2020-09-16 13:47:16