| 1. |
- Byström, Roberth, 1971-
(författare)
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SOD1´s Law : An Investigation of ALS Provoking Properties in SOD1
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are the most important molecules in the cell since they take care of most of the biological functions which resemble life. To ensure that everything is working properly the cell has a rigorous control system to monitor the proper function of its proteins and sends old or dysfunctional proteins for degradation. Unfortunately, this system sometimes fails and the once so vital proteins start to misbehave or to accumulate and in the worst case scenario these undesired processes cause the death of their host. One example is Amyotrophic Lateral Sclerosis (ALS); a progressive and always fatal neurodegenerative disorder that is proposed to derive from accumulation of aberrant proteins. Over 140 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) are linked to ALS. The key event in SOD1 associated ALS seems to be the pathological formation of toxic protein aggregates as a result of initially unfolded or partly structured SOD1-mutants. Here, we have compared the folding behaviour of a set of ALS associated SOD1 mutants. Based on our findings we propose that SOD1 mediated ALS can be triggered by a decrease in protein stability but also by mutations which reduce the net charge of the protein. Both findings are in good agreement with the hypothesis for protein aggregation. SOD1 has also been found to be able to interact with mitochondrial membranes and SOD1 inclusions have been detected in the inter-membrane space of mitochondria originating from the spinal cord. The obvious question then arose; does the misfolding and aggregation of SOD1 involve erroneous interactions with membranes? Here, we could show that there is an electrostatically driven interaction between the reduced apo SOD1 protein including ALS associated SOD1-mutants and charged lipid membrane surfaces. This association process changes the secondary structures of these mutants in a way quite different from the situation found in membrane free aqueous environment. However, the result show that mutants interact with charged lipid vesicles to lesser extent than wildtype SOD1. This opposes the correlation between decreased SOD1 stability and disease progression. We therefore suggest that the observed interaction is not a primary cause in the ALS mechanism.
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| 2. |
- Vallina Estrada, Eloy, 1993-
(författare)
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Navigating the Cellular Crowd : Physicochemical Properties of Protein Surfaces as Evolved Interaction Guides
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cellular interior is characterised by high concentrations of macromolecules. Compared with dilute conditions, the crowd modifies proteins' ability to fold, diffuse and, ultimately, carry out their biological functions. Cellular fitness depends on ensuring an adequate balance between interactivity and diffusivity.In this thesis, I discuss how a colloidal description of the cell highlights the central role of electrostatics in protein surface optimisation. By recognising that the modulation of protein-protein interactions concerns the whole proteome, I map the physicochemical preferences of cellular organisms across taxonomic and ecological divisions. Moreover, I propose that all surface residues participate in tuning protein interactions to the correct affinity, within a continuum that spans several orders of magnitude. Finally, I turn to horizontally spreading inteins to gauge the strength of the selective pressures acting on protein surfaces.
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| 3. |
- Österlund, Nicklas, 1992-
(författare)
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Capturing transient peptide assemblies associated with Alzheimer's disease : Native mass spectrometry studies of amyloid-β oligomerization
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Correct folding of proteins is essential for maintaining a functional living cell. Misfolding and aggregation of proteins, where non-native intermolecular interactions form large and highly ordered amyloid aggregates with low free energy, is hence associated with multiple diseases. One example is Alzheimer’s disease (AD) where the Amyloid-β (Aβ) peptide aggregates into amyloid fibrils, which deposit as neuritic plaques in the brains of AD patients. Nucleation of amyloid fibrils takes place via formation of smaller pre-nucleation clusters, so-called oligomers, which are considered to be especially toxic and are therefore potentially important in AD pathology. Detailed mechanistic molecular knowledge of Aβ aggregation is important for design of AD treatments that target these processes. The oligomeric species are however challenging to study experimentally due to their low abundance and high polydispersity. Aβ oligomers are in this thesis studied under controlled in vitro conditions using bottom-up biophysics. Highly pure recombinant Aβ peptides are studied, primarily using native ion-mobility mass spectrometry, to monitor the spontaneous formation of oligomers in aqueous solution. Mass spectrometry is capable of resolving individual oligomeric states, while ion mobility provides low-resolution structure information. This is complemented with other biophysical techniques, as well as theoretical modeling. The oligomers are also studied upon modulating intrinsic factors, such as peptide length and sequence, or extrinsic factors, such as the chemical environment. Interactions with two important biological interaction partners are studied: chaperone proteins and cell membranes. We show how Aβ oligomers assemble, and form extended structures which may be linked to continued growth into amyloid fibrils. We also show how different amyloid chaperone proteins interact with growing aggregates, which modifies and delays the aggregation process. These interactions are shown to depend on specific sequence-motifs in the chaperones and client peptides. Membrane-mimicking micelles are on the other hand able to stabilize globular compact forms of the Aβ oligomers and to inhibit the formation of extended structures which nucleate into amyloid fibrils. This may contribute to enrichment of toxic species in vivo. Interactions with membrane-mimicking systems are shown to be highly dependent on both the Aβ peptide isoform and the properties of the membrane environment, such as headgroup charges. It is also demonstrated how addition of a designed small peptide construct can inhibit formation of Aβ oligomers in the membrane environment.
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| 4. |
- Kurnik, Martin, 1980-
(författare)
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Protein folding without loops and charges
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Going down the folding funnel, proteins may sample a wide variety of conformations, some being outright detrimental to the organism. Yet, the vast majority of polypeptide molecules avoid such pitfalls. Not only do they reach the native minimum of the energy landscape; they do so via blazingly fast, biased, routes. This specificity and speed is remarkable, as the surrounding solution is filled to the brim with other molecules that could potentially interact with the protein and in doing so stabilise non-native, potentially toxic, conformations. How such incidents are avoided while maintaining native structure and function is not understood. This doctoral thesis argues that protein structure and function can be separated in the folding code of natural protein sequences by use of multiple partly uncoupled factors that act in a concerted fashion. More specifically, we demonstrate that: i) Evolutionarily conserved functional and regulatory elements can be excised from a present day protein, leaving behind an independently folded protein scaffold. This suggests that the dichotomy between functional and structural elements can be preserved during the course of protein evolution. ii) The ubiquitous charges on soluble protein surfaces are not required for protein folding in biologically relevant timescales, but are critical to intermolecular interaction. Monomer folding can be driven by hydrophobicity and hydrogen bonding alone, while functional and structural intermolecular interaction depends on the relative positions of charges that are not required for the native bias inherent to the folding mechanism. It is possible that such uncoupling reduces the probability of evolutionary clashes between fold and function. Without such a balancing mechanism, functional evolution might pull the carpet from under the feet of structural integrity, and vice versa. These findings have implications for both de novo protein design and the molecular mechanisms behind diseases caused by protein misfolding.
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| 5. |
- Lang, Lisa, 1973-
(författare)
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SOD1 Aggregation : Relevance of thermodynamic stability
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism.Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur. Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.
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| 6. |
- Leeb, Sarah, 1989-
(författare)
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How transient interactions in the crowded cytosol affect protein mobility and stability
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most biochemical reactions have evolved in crowded intracellular environments. However, the complexity of intracellular environments is often neglected in structural or functional studies of proteins. In these cases, reactions involving proteins are deliberately separated from the perturbations of co-solutes in order to simplify data acquisition and interpretation. Having acquired an enormous body of knowledge under these simplified dilute-buffer conditions, methodological progress of the past two decades has made the study of proteins inside living cells increasingly accessible and concomitantly kindled an interest to investigate proteins in their native habitat. Naturally, major questions that arose were to what extent ubiquitous transient interactions alter protein structure, function and thermodynamics and, not least, what role protein surfaces and their physicochemical properties play in determining the frequency and duration of these diffusive encounters.By looking at the rotational-tumbling rates of three structurally well-characterized proteins in live cells with nuclear magnetic resonance (NMR) relaxation, we expand on previous research performed in the bacterium Escherichia coli and establish the physicochemical principles that determine diffusive interactions in the mammalian cytosol of the human ovarian cancer cell line A2780. Just as in E. coli, net charge is the dominating factor in regulating protein interactivity, albeit with the impact on rotational retardation greatly diminished. We ascribe this to the generally lower macromolecular concentrations in the eukaryotic cytosol, and put forward a hypothesis in which less stringent rules regarding protein surface decoration in eukaryotes could have facilitated the development of multi-cellular organisms. Furthermore, by developing a model where a distribution of differently sized interaction partners is taken into account when examining rotational retardation, we reconcile transverse and longitudinal in-cell relaxation with theory, and are able to estimate the populations of the bound and free form of a set of reporter proteins. Looking at the populations of bound protein instead of a mean-field rotational retardation finally allows us to re-assess the guiding rules behind diffusive cytosolic interactions. Last, we outline a putative mechanism behind the in-cell destabilization of a variant of Superoxide dismutase 1 (SOD1barrel). By mimicking generic poly-anionic intracellular co-solutes with poly-acetic acid (NaPAc1200), we identify the positively charged N-terminal portion of the unfolded form of the protein as the interaction site with the highest affinity. Further examining the unfolded ensemble of SOD1barrel with a mutationally destabilized variant reveals a compact state, that remains almost unchanged upon binding to NaPAc1200. This suggests that NaPAc1200-mediated destabilization occurs mainly through mass action, in full accord with the postulated mechanism for in-cell protein destabilization.
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| 7. |
- Leinartaité, Lina, 1982-
(författare)
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Zinc in folding and misfolding of SOD1 : Implications for ALS
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing degeneration of upper and lower motor neurons. Most ALS cases are sporadic; only 6% are associated with mutations in Cu, Zn superoxide dismutase (SOD1). It is believed, however, that sporadic and familiar forms of ALS share a common mechanism, where SOD1 plays an important role: SOD1 knockout mice do not develop ALS, whereas the overexpression of human SOD1 in mice produces ALS-like symptoms. Increasing evidence suggest that the SOD1 structure gains cytotoxic properties, but detailed description of the toxic species is missing. This thesis work is focused on understanding how structural and dynamic properties of SOD1 change along its folding free-energy landscape and indicates the structural hot-spots from where the cytotoxic species may originate. Thus, binding of the zinc controls folding, stability and turnover of SOD1: (i) miscoordination of Zn2+ by the Cu-ligands speeds up folding of the SOD1 core structure, however, it stabilizes SOD1 in a state where both active-site loops IV and VII are unfolded, (ii) coordination of Zn2+ in the Zn-site, induces the folding of loop VII and stabilizes the native and functional fold of both active-site loops and (iii) the tremendous stability gain due to Zn-site metallation corresponds to a folded state’s lifetime of > 100 years, thus the cellular lifetime of SOD1 is likely controlled by Zn2+ release, which again is coupled to opening of active-site loops. Hence the active-site loops IV and VII stand out as critical and floppy parts of the SOD1 structure. Moreover, a number of ALS-associated mutations, benign to apo-SOD1 stability, are shown here to affect integrity of active-site loops in holo-SOD1, which, in turn, increases population of SOD1 species with these loops disorganized. Finally, the close relation between SOD1 and Zn2+ can also act in the reverse direction: a perturbed folding free-energy landscape of SOD1 can disturb Zn2+ homeostasis.
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| 8. |
- Mu, Xin, 1987-
(författare)
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Protein stability and mobility in live cells : Revelation of the intracellular diffusive interaction organization mechanisms
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biochemical processes inside living cells take place in a confined and highly crowded environment. As such, macromolecular crowding, one of the most important physicochemical properties of cytoplasm, is an essential element of cell physiology. It not only gives rise to steric repulsion, but also promotes non-specific, transient, interactions (referred to as diffusive interactions) between molecules. Since diffusive interactions are a key way to achieving a highly organized intracellular environment, without such interactions, the cell is just “a bag of molecules”. Therefore, understanding how diffusive interactions modulate protein behavior in live cells is of fundamental importance for revealing the mechanisms of molecular recognition, as well as for understanding the cause of protein misfolding diseases.This thesis focuses on how macromolecular crowding influences the stability and diffusive motions of proteins within living cells by modulating their diffusive interactions. First, we investigated the thermal stability of superoxide dismutase 1 (SOD1), a protein involved in the development of familial amyotrophic lateral sclerosis (ALS), in mammalian and E. coli cells. Intriguingly, the major thermodynamic consequence of macromolecular crowding is due not only to conventional steric repulsions, but primarily to the detailed chemical nature of the diffusive protein interactions in live cells. Secondly, we presented a mutational study of how these diffusive interactions influence the rotation of proteins in the mammalian and bacterial cytosol. The result is a quantitative description of the physicochemical code for the intracellular protein motion, showing that it depends critically on the surface details of protein and the type of the host cell as well. Thirdly, we characterized the impact of intracellular protein concentration by altering the volume of E. coli cells by osmotic shock. The results obtained show that the intracellular diffusion of proteins is not determined by the chemical properties of the protein surface alone, but also by the frequency of concentration-dependent encounters. Moreover, it appears that eukaryotes and bacteria have achieved fidelity of biological processes through different evolutionary strategies. Overall, these observations have numerous implications for both functional protein design and deciphering the evolution of the surface characteristics of proteins.Subsequently, we attempted to shed new light on the Hofmeister series, using protein-folding kinetics as observable. The results indicate that the Hofmeister series cannot be explained entirely by the traditional Kosmotropes/Chaotropes classification. Strong hetero-ion pairing cannot be ignored when trying to understand the effects of salts on protein salting-in and salting-out behaviors.
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| 9. |
- Oskarsson, Marie, 1982-
(författare)
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Islet amyloid polypeptide (IAPP) in Type 2 diabetes and Alzheimer disease
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The misfolding and aggregation of the beta cell hormone islet amyloid polypeptide (IAPP) into amyloid fibrils is the main pathological finding in islets of Langerhans in type 2 diabetes. Pathological assemblies of IAPP are cytotoxic and believed to contribute to the loss of insulin-producing beta cells. Changes in the microenvironment that could trigger the aggregation of IAPP are largely unknown. So is the possibility that islet amyloid can spread within or between tissues. The present thesis have explored the roles of glycosaminoglycan heparan sulfate (HS) and the novel anti-amyloid chaperone Bri2 BRICHOS domain in the assembly of IAPP amyloid and cytotoxic IAPP aggregates. Furthermore, cross-seeding as a molecular interaction between the observed connection of type 2 diabetes and Alzheimer disease has been examined.The N-terminal region of IAPP was required for binding to HS structures and induction of binding promoted amyloid formation. Interference in the HS-IAPP interaction by heparanase degradation of HS or by introducing short, soluble HS-structure fragments reduced amyloid deposition in cultured islets. Cytotoxicity induced by extracellular, aggregating IAPP was mediated via interactions with cell-surface HS. This suggests that HS plays an important role in islet amyloid deposition and associated toxicity.BRICHOS domain containing protein Bri2 was highly expressed in human beta cells and colocalized with IAPP intracellularly and in islet amyloid deposits. The BRICHOS domain effectively attenuated both IAPP amyloid formation and IAPP-induced cytotoxicity. These results propose Bri2 BRICHOS as a novel chaperone preventing IAPP aggregation in beta cells.The intravenous injection of IAPP, proIAPP or amyloid-β (Aβ) fibrils enhanced islet amyloidosis in transgenic human IAPP mice, demonstrating that both homologous- and heterologous seeding of islet amyloid can occur in vivo. IAPP colocalized with Aβ in brain amyloid from AD patients, and AD patients diagnosed with T2D displayed increased proportions of neuritic plaques, the more pathogenic plaque subtype.In conclusion, both IAPP amyloid formation and the cytotoxic effects of IAPP is dependent on interactions with HS whereas interactions with Bri2 BRICHOS is protective. Cross-seeding between Aβ and IAPP can occur in vivo and the two peptides colocalize in brain amyloid in AD patients.
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| 10. |
- Sörensen, Therese, 1987-
(författare)
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Diffusive interactions play an important role in protein stability and mobility : Investigations of the intracellular milieu using in-cell NMR
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are crucial for all cellular life. Every signal received by a cell, and every response to it, is mediated by proteins. Inside cells, these proteins diffusively sample each other’s surfaces, as they travel through the cytoplasm in search of their specific interaction partners. In order to carry out their function, proteins need to navigate this net negatively charged and highly crowded milieu without getting stuck with undesirable partners. How do they achieve this?Previously published data has shown that the bacterial cytoplasm is governed by physicochemical restrictions: There is a net charge interval within which proteins remain soluble. Meaning, if a protein is too positively charged, it will get stuck to the surrounding molecules. If it is too negatively charged, the intracellular mobility approaches that in water, potentially reducing the chance of the protein finding its functional partner. Using in-cell NMR, we have shown that similar charge-based rules govern the molecular mobility inside human cells. The less crowded human cytoplasm does, however, seem more forgiving than the bacterial counterpart, as proteins that experience restricted mobility inside bacteria seem to move freely inside human cells.The human and bacterial cytoplasm both have a destabilising effect on the ALS-associated ROS scavenger Superoxide Dismutase 1 (SOD1). Our results show that: Stabilised by electrostatic interactions between the positively charged N-terminal and the negatively charged contents of the cytoplasm, the folding equilibrium is shifted towards the unfolded state. Additionally, in the absence of metals, native metal-coordinating surface-exposed histidine residues also contribute to the intracellular destabilisation of SOD1.Finally, the unfolded state of SOD1 has been characterised in the absence of chemical denaturants. We show that the unfolded state is more compact than previously anticipated. We hypothesise that the increased compactness is caused by the pre-formation of long-range native-like contacts. This implies that: Not only does the primary structure contain the information required for folding, it also contains information on how the unfolded state needs to organise itself to increase the probability of successful folding.
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| 11. |
- Yang, Fan, 1988-
(författare)
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Biophysical chemistry of the ALS-associated protein SOD1 : Implications for folding, aggregation and in-cell behaviour
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biophysical chemistry deals with the structural behavior, properties and molecular function of biological macromolecules. A long-standing challenge is here to establish how these macromolecular features change upon transfer from simplified conditions in vitro to the crowded and molecularly complex environment of live cells. This thesis focuses on establishing a general overview of the structural behavior and interaction properties of the ALS-associated protein superoxide dismutase 1 (SOD1) in its natural cellular environment. Importantly, SOD1 constitutes also a multifaceted model system for the yet poorly understood mechanism of protein-aggregation disease, since it is readily amenable to protein-engineering analysis. The focus is on (i) SOD1 folding, (ii) the modulation of the SOD1 properties induced by intracellular interactions and (iii) the process of SOD1 fibrillation, all of which central to the understanding of the ALS disease mechanism. First, we investigate the biophysical role of the disordered catalytic loops in the apoSOD1 monomer, what is identified as the primary aggregation precursor. The results show that these loops play a pivotal role in modulation the apoSOD1 stability due to the generic Flory-entropy penalty, shedding new light to why this species is biased to be aggregation prone. Second, we target the diffusive interactions between SOD1 and the crowded intracellular environment by in-cell NMR. Our findings are that both the rotational tumbling and in-cell stability are controlled by basic physicochemical rules relating to the SOD1 surface properties. Finally, we analyze the kinetics of the SOD1-aggregation behavior in vitro. The observations confirm that the disordered SOD1 loops indeed accelerate the aggregation process because of their penalty to the apo state stability and show, additionally, that they influence the fibril stability.The physicochemical cues exposed by this thesis work provide not only fundamental clues to our understanding of protein properties, but shed also new light on disease-promoting properties ALS-associated protein SOD1.
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| 12. |
- Haglund, Ellinor, 1978-
(författare)
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Folding of the Ribosomal protein S6 : The role of sequence connectivity, overlapping foldons, and parallel pathways
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To investigate how protein folding is affected by sequence connectivity five topological variants of the ribosomal protein S6 were constructed through circular permutation. In these constructs, the chain connectivity (i.e. the order of secondary-structure elements) is changed without changing the native-state topology. The effects of the permutations on the folding process were then characterised by φ-value analysis, which estimates the extent of contact formations in the transition-state ensemble. The results show that the folding nuclei of the wild-type and permutant proteins comprises a common motif of one α-helix docking against two β-sheets, i.e. the minimal structure for folding. However, this motif is recruited in different parts of the S6 structure depending on the permutation, either in the α1 or α2 half of the protein. This minimal structure is not unique for S6 but can also be seen in other proteins. As an effect of the dual nucleation possibilities, the transition-state changes describe a competition between two parallel pathways, which both include the central β-stand 1. This strand constitutes thus a structural overlap between the two competing nuclei. As similar overlap between competing nuclei is also seen in other proteins, I hypothesise that the coupling of several small nuclei into extended ‘super nuclei’ represents a general principle for propagating folding cooperativity across large structural distances. Moreover, I demonstrate by NMR analysis that the existence of multiple folding nuclei renders the H/D-exchange kinetics independent of the folding pathway.
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| 13. |
- Hedberg, Linda, 1976-
(författare)
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The birth and growth of the protein folding nucleus : Studies of protein folding focused on critical contacts, topology and ionic interactions
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are among the most complex molecules in the cell and they play a major role in life itself. The complexity is not restricted to just structure and function, but also embraces the protein folding reaction. Within the field of protein folding, the focus of this thesis is on the features of the folding transition state in terms of growing contacts, common nucleation motifs and the contribution of charged residues to stability and folding kinetics. During the resent decade, the importance of a certain residue in structure formation has been deduced from Φ-value analysis. As a complement to Φ-value analysis, I present how scatter in a Hammond plot is related to site-specific information of contact formation, Φ´(βTS), and this new formalism was experimentally tested on the protein L23. The results show that the contacts with highest Φ growth at the barrier top were distributed like a second layer outside the folding nucleus. This contact layer is the critical interactions needed to be formed to overcome the entropic barrier. Furthermore, the nature of the folding nucleus has been shown to be very similar among proteins with homologous structures and, in the split β-α-β family the proteins favour a two-strand-helix motif. Here I show that the two-strand-helix motif is also present in the ribosomal protein S6 from A. aeolicus even though the nucleation and core composition of this protein differ from other related structure-homologues. In contrast to nucleation and contact growth, which are events driven by the hydrophobic effect, my most recent work is focused on electrostatic effects. By pH titration and protein engineering the charge content of S6 from T. thermophilus was altered and the results show that the charged groups at the protein surface might not be crucial for protein stability but, indeed, have impact on folding kinetics. Furthermore, by site-specific removal of all acidic groups the entire pH dependence of protein stability was depleted.
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| 14. |
- Karlsson, Martin, 1965-
(författare)
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Protein engineering for biophysical studies of protein folding, stability and surface interactions
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The subject of this thesis can be split into two parts, one that is dealing with the stability and stabilization of proteins on its own merits and a second part that deals with the protein adsorption orientation on solid surfaces and how the stability of a protein influences the behavior at a solid/liquid interface. Thus, the common denominator and focus have been on protein stability. Molecular modeling and site-directed mutagenesis tools have been used to engineer a protein, human carbonic anhydrase II (HCA II), for these studies. The effects of these mutations on stability and surface interactions have then been studied by biophysical methods.Stabilization of HCA II by an engineered disulfide bridge: To find a way to stabilize the enzyme HCA II, homology modeling against the related and unusually stable carbonic anhydrase from Neisseria gonorrhoeae (NGCA) was performed. We were able to successfully utilize the homology modeling to graft a disulfide bridge from NGCA into the human enzyme. The disulfide bond was not formed spontaneously, but would only form after a prolonged exposure to oxidizing agents. However, formation of the disulfide bridge led to a dramatic stabilization of the native conformation.Accelerated formation of the disulfide bridge: It was found that it is the conformationally restrained localization of the introduced cysteines that is the reason that the protein is expressed in the reduced state and is not readily oxidized. However, upon exposure to low concentrations of denaturant, corresponding to the lower part of the denaturation curve for the first unfolding transition of the reduced state, there was a striking increase of the oxidation rate of correctly formed disulfide bridges. This provides a method for creating the oxidized disulfide variant of proteins, with engineered cysteines in the interior of proteins, which would otherwise not be formed within an acceptable time span.Refolding studies of stabilized variant of HCA II: The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition with suppression of the otherwise stable equilibrium. molten-globule intermediate, which normally is very prone to aggregation. Stopped-flow measurements also showed that the population of the transiently occurring molten globule was suppressed during refolding. This circumnavigation of misfolding traps and intermediates led to a markedly lowered tendency for aggregation and to significantly higher reactivation yields upon refolding of the fully denatured protein.Correlation between protein stability and surface induced denaturation: Negatively charged silica nanoparticles were used in order to determine the influence of protein stability on the denaturation rate upon adsorption. Various destabilized mutants were produced by site-directed mutagenesis of amino acids located in the interior of the protein. The silica nanoparticles induced a molten globule-like state in all of the variants. All protein variants initially adsorbed to the particles, and subsequently underwent conformational rearrangements in a stepwise manner. This study also showed that a decrease in the global stability of the protein is strongly correlated to increased rates of conformational change upon adsorption to the surface.Determination of protein adsorption orientation: By site-directed labeling fluorescent probes were specifically introduced on the surface of HCA II and it was shown, for the first time, that it is possible to specifically determine the orientation of an adsorbed protein in the native state to a surface (silica nanoparticles). By this approach it was possible to clearly demonstrate that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein and, furthermore, that the adsorption direction is strongly pH-dependent.Reduction of irreversible protein adsorption by protein stabilization: The strong correlation between decreased stability and increased rates of conformational changes of the protein upon adsorption to surfaces initiated yet another surface study. Three variants of HCA IT with lower, the same, and higher stability than the wild-type protein were monitored by surface plasmon resonance upon adsorption to and desorption from surfaces with fundamentally different properties. Regardless of the nature of the surface there were correlations between (i) the protein stability and kinetics of adsorption with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability, demonstrating that protein engineering for increased stability can be used to reduce irreversible protein adsorption.
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