| 1. |
- Bucak, S, et al.
(författare)
-
Peptide Nanotube Nematic Phase.
- 2009
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 25, s. 4262-4265
-
Tidskriftsartikel (refereegranskat)abstract
- The self-assembly of the trifluoroacetate salt of the short peptide (ala)(6)-lys (A(6)K) in water has been investigated by cryo-transmission electron microscopy and small-angle X-ray scattering. For concentrations below ca. 12%, the peptide does not self-assemble but forms a molecularly dispersed solution. Above this critical concentration, however, A(6)K self-assembles into several-micrometer-long hollow nanotubes with a monodisperse cross-sectional radius of 26 nm. Because the peptides carry a positive charge, the nanotubes are charge-stabilized. Because of the very large aspect ratio, the tubes form an ordered phase that presumably is nematic.
|
|
| 2. |
- Nasir, Irem, et al.
(författare)
-
Fluorescent Filter-Trap Assay for Amyloid Fibril Formation Kinetics in Complex Solutions.
- 2015
-
Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 6:8, s. 1436-1444
-
Tidskriftsartikel (refereegranskat)abstract
- Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (t1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration.
|
|
| 3. |
- Nasir, Irem
(författare)
-
Fluorescent Probes and Protein Misfolding: Methods and Applications
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
|
|
| 4. |
- Nasir, Irem, et al.
(författare)
-
High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.
- 2015
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:8
-
Tidskriftsartikel (refereegranskat)abstract
- The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions.
|
|
| 5. |
- Nasir, Irem, 1987, et al.
(författare)
-
Ratiometric Single-Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins
- 2020
-
Ingår i: Current protocols in chemical biology. - : Wiley. - 2160-4762 .- 2160-4762. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid-liquid phase separation. The so-called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein "structure-function paradigm," as their functions do not necessarily involve well-defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single-molecule fluorescence methods can overcome this ensemble-average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single-molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in-depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel.
|
|
| 6. |
- Nasir, Irem, 1987, et al.
(författare)
-
Single-molecule fluorescence studies of intrinsically disordered proteins and liquid phase separation
- 2019
-
Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1878-1454 .- 1570-9639. ; 1867:10, s. 980-987
-
Forskningsöversikt (refereegranskat)abstract
- Intrinsically disordered proteins (IDPs) are ubiquitous in proteomes and serve in a range of cellular functions including signaling, regulation, transport and enzyme function. IDP misfunction and aggregation are also associated with several diseases including neurodegenerative diseases and cancer. During the past decade, single-molecule methods have become popular for detailed biophysical and structural studies of these complex proteins. This work has included recent applications to cellular liquid-liquid phase separation (LLPS), relevant for functional dynamics of membraneless organelles such as the nucleolus and stress granules. In this concise review, we cover the conceptual motivations for development and application of single-molecule fluorescence methods for such IDP studies. We follow with a few key examples of systems and biophysical problems that have been addressed, and conclude with thoughts for emerging and future directions.
|
|
| 7. |
- Nasir, Irem, et al.
(författare)
-
Size and surface chemistry of nanoparticles lead to a variant behavior in the unfolding dynamics of human carbonic anhydrase.
- 2015
-
Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 7:41, s. 17504-17515
-
Tidskriftsartikel (refereegranskat)abstract
- The adsorption induced conformational changes of human carbonic anhydrase I (HCAi) and pseudo wild type human carbonic anhydrase II truncated at the 17th residue at the N-terminus (trHCAii) were studied in presence of nanoparticles of different sizes and polarities. Isothermal titration calorimetry (ITC) studies showed that the binding to apolar surfaces is affected by the nanoparticle size in combination with the inherent protein stability. 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence revealed that HCAs adsorb to both hydrophilic and hydrophobic surfaces, however the dynamics of the unfolding at the nanoparticle surfaces drastically vary with the polarity. The size of the nanoparticles has opposite effects depending on the polarity of the nanoparticle surface. The apolar nanoparticles induce seconds timescale structural rearrangements whereas polar nanoparticles induce hours timescale structural rearrangements on the same charged HCA variant. Here, a simple model is proposed where the difference in the timescales of adsorption is correlated with the energy barriers for initial docking and structural rearrangements which are firmly regulated by the surface polarity. Near-UV circular dichorism (CD) further supports that both protein variants undergo structural rearrangements at the nanoparticle surfaces regardless of being "hard" or "soft". However, the conformational changes induced by the apolar surfaces differ for each HCA isoform and diverge from the previously reported effect of silica nanoparticles.
|
|
| 8. |
- Sanagavarapu, Kalyani, et al.
(författare)
-
A method of predicting the in vitro fibril formation propensity of Aβ40 mutants based on their inclusion body levels in E. coli
- 2019
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9:1
-
Tidskriftsartikel (refereegranskat)abstract
- Overexpression of recombinant proteins in bacteria may lead to their aggregation and deposition in inclusion bodies. Since the conformational properties of proteins in inclusion bodies exhibit many of the characteristics typical of amyloid fibrils. Based on these findings, we hypothesize that the rate at which proteins form amyloid fibrils may be predicted from their propensity to form inclusion bodies. To establish a method based on this concept, we first measured by SDS-PAGE and confocal microscopy the level of inclusion bodies in E. coli cells overexpressing the 40-residue amyloid-beta peptide, Aβ40, wild-type and 24 charge mutants. We then compared these results with a number of existing computational aggregation propensity predictors as well as the rates of aggregation measured in vitro for selected mutants. Our results show a strong correlation between the level of inclusion body formation and aggregation propensity, thus demonstrating the power of this approach and its value in identifying factors modulating aggregation kinetics.
|
|
