Browsing by Author "Wittung-Stafshede, Pernilla"

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    Copper as a biological yin-yang element: Structural dynamics, protein -protein interactions and transfer mechanisms of copper transport proteins
    (2009) Rodriguez-Granillo, Agustina; Wittung-Stafshede, Pernilla
    Copper (Cu) can be considered as a biological yin-yang element, because it is essential but toxic at the same time. To manage this paradox, cells have evolved complex molecular Cu transport pathways, in which Cu chaperones bind and shuttle Cu to specific intracellular targets. In humans, Atox1 delivers Cu(I) to the metal-binding domains (MBDs) of Cu-ATPases, the Menkes and Wilson disease proteins, for subsequent incorporation into cuproenzymes. Both metallochaperones and target MBDs adopt the same ferredoxin-like fold and bind Cu(I) via two Cys residues in a conserved motif. In this thesis, we have employed a wide selection of state-of-the-art computational schemes, including quantum mechanics and molecular mechanics methodologies in combination with molecular dynamics simulations, to broaden our understanding on the structure-function relationships of Cu chaperones, MBDs, and their interactions. This work reports on a thorough study of the structural dynamics, Cu-binding properties, protein-protein interactions and Cu-transfer mechanisms of key Cu transport proteins, and how conserved residues modulate these properties. We found that residues framing the Cu loop have evolved differently in prokaryotic and eukaryotic Cu chaperones to tune the flexibility and provide an optimal stabilization of the Cu loop. Some of these residues are also key for metallochaperone-MBD interactions and subsequent Cu(I) transfer. We further found that the MBDs are not equivalent at the molecular level, and propose that backbone flexibility together with electrostatic complementarity are important factors to guide Atox1 interactions. We propose that Atox1 interacts with its partner MBDs via a "weak" interface that can be disrupted by at least one substitution in Atox1. Finally, we have elucidated for the first time the Cu(I) transfer mechanism from holo-Atox1 to an apo-MBD, and propose that the reaction proceeds with the existence of two trigonal intermediates. Our results suggest that the reaction is kinetically feasible but not energetically favorable, pointing to the apparent absence of a thermodynamic gradient for Cu(I) transfer. The structural, dynamic, thermodynamic and mechanistic details obtained here with atomic resolution are difficult to obtain by in vitro experiments, and can be used both as a complement to experiments and as predictive tools for functional insights.
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    Effects of macromolecular crowding and small ions on the folding, structure, and stability of Desulfovibrio desufuricans flavodoxin
    (2010) Stagg, Loren; Wittung-Stafshede, Pernilla
    The intracellular environment in which most proteins fold and function contains a range of biomolecules that results in significant volume exclusion, thus contrasting to the dilute buffer conditions common to most in vitro studies. In addition to intracellular macromolecular crowding, cells are ionic in nature, and although the Hofmeister series of ions has its origin in a work from 1888, much is still unclear concerning how small, charged ions affect protein properties. This thesis summarizes in vitro work assessing the effects of macromolecular crowding and small ions on the biophysical properties of a model protein -- Desulfovibrio desulfuricans flavodoxin. Flavodoxin is a small (15.7 kDa), single domain, cytoplasmic protein with alpha-helical and parallel beta-sheet secondary structural elements arranged in one of the five most common protein folds (the flavodoxin-like fold). Using a range of biophysical/spectroscopic methods (e.g., circular dichroism (CD), fluorescence, calorimetry, stopped-flow mixing) along with synthetic crowding agents (e.g., Ficoll and dextran), I have found that macromolecular crowding increases the secondary structural content of folded flavodoxin (toward that found in the crystal structure), increases flavodoxin thermal stability, and affects both the accumulation of a misfolded intermediate and the rate of proper protein folding. Collaborative in silico simulations employing Go-like modeling of apoflavodoxin in the presence of large, inert crowding agents agrees with my in vitro work and provides structural and mechanistic information with residue-specific resolution. We also found that small cations and anions in physiologically relevant concentrations (≤ 250 mM) increase flavodoxin thermal stability significantly. Both cations and anions in higher concentrations (300 mM-.75 M) affect oppositely charged proteins similarly suggesting that surface electrostatic charge plays only a minor role in mediating ionic effects on protein thermal stability. At all ion concentrations, ionic effects on protein stability are correlated to ion hydration (and thus the Hofmeister series). Our work suggests a dominant role for the peptide bond in coordinating ions at higher concentrations. This thesis work suggests that the crowded and ionic nature of the intracellular milieu can elicit changes to the structure, dynamics, stability, and folding mechanism of proteins which may not be captured in vitro using dilute buffer conditions.
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    Exploring the envelope of life: Folding and assembly reactions of hyper-thermostable co-chaperonin protein 10 from Aquifex aeolicus
    (2007) Luke, Kathryn A.; Wittung-Stafshede, Pernilla
    The co-chaperonin protein 10 (cpn10) is a heptameric ring-shaped protein present in most organisms. It works in conjunction with the chaperonin protein 60 (cpn60) in an ATP-dependent process to assist folding a range of substrate polypeptides. Cpn10 from the hyper-thermophilic bacterium Aquifex aeolicus (Aacpn10) contains a 25-residue C-terminal extension in each monomer, not found in any other cpn10 protein. Both Aacpn10 and a mutant where the tail has been removed (Aacpn10del-25) adopt heptameric structures with similar thermal and chemical stabilities. In addition, the equilibrium and kinetic unfolding/dissociation and refolding/reassembly reactions are not affected by the presence or absence of the tail. The presence of the tail, however, increases the affinity between the subunits in the heptamer and limits the formation of ordered aggregates of the heptamers at high temperatures and high protein concentrations. Comparative studies on mesostable cpn10 heptamers from human mitochondria (hmcpn10) and Escherichia coli (GroES) reveal that the extreme stability of Aacpn10 originates from increased stability of individual monomers. The stability profile (i.e., the correlation between free energy and temperature) for Aacpn10 is shifted upwards (i.e., higher stability at each temperature) and to the right (i.e., maximum stability at higher temperature) as compared to that of GroES. This is the first thermodynamic analysis of how hyper-thermostability is achieved in an oligomeric protein system.
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    Managing the copper paradox: Protein stability, copper-binding, and inter-protein interactions of copper chaperones
    (2010) Hussain, Faiza; Wittung-Stafshede, Pernilla
    To minimize copper (Cu) toxicity, organisms have evolved Cu transport pathways involving soluble metallochaperones that bind, transport, and deliver Cu+ to specific partner proteins, such as Cu-ATPases. The human Cu chaperone, Atox1, delivers Cu to the metal-binding domains of Menkes (MNK) and Wilson (WND) disease proteins that are Cu-ATPases in the Golgi network that transfer Cu to cuproenzymes (e.g., ceruloplasmin) that traverse the Golgi lumen. The metal binding motif, MetX1CysXXCys, and the ferredoxin-like fold appear conserved in both cytoplasmic Cu chaperones and the cytoplasmic metal-binding domains of the target Cu-ATPases from different organisms. The work reported here provides a basic understanding of in vitro holo- and apo-protein stability, Cu-dissociation mechanisms, and donor-acceptor interactions of key copper transport chaperones. Studies were conducted on purified protein variants using circular dichroism, fluorescence, and absorbance methods in equilibrium and time-resolved modes. We developed a kinetic assay to determine the Cu-dissociation mechanism of these proteins and a near-UV CD method for monitoring interactions between Atox1 and WND domains to complement NMR measurements and computer simulations. Despite the conservation of the overall structural fold, the chaperones Atox1 and its bacterial homolog, CopZ, and the metal-binding domains of WND, W2 and W4, have variable chemical and thermal stability in vitro. The role of residues proximal to the metal-binding site was determined using Atox1 as a prototypical Cu chaperone. Met10 is essential for structural stability of Atox1. Thr11 (position X1) seems to be conserved, not for integrity of protein structure, but for facilitating metal exchange between Atox1 and a receptor domain. The structural proximity of the charged side-chain of Lys60 neutralizes the Cu-thiolate center in Atox1. Replacement of Lys60 with an Ala or Tyr results in a higher rate and extent of loss of the metal to small molecule chelator, BCA, than those for wtAtox1. Lys60 also provides electrostatic interactions crucial for Atox1 interaction with W4. Thus, each proximal residue contributes to fine-tuning copper binding and its release mechanism to both the non-physiological Cu chelator, BCA, and the physiological acceptor of the WND protein, W4. Our new kinetic and spectral assays provide a comprehensive in vitro experimental platform for more advanced future mechanistic and kinetic studies.
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    Roles of specific and non-specific interactions in folding of beta-sheet and alpha-helical protein model systems
    (2008) Perham, Michael F.; Wittung-Stafshede, Pernilla
    To get a comprehensive understanding of protein folding, the structural complexity of many proteins as well as the properties of the cellular milieu must be considered. For oligomeric proteins, not only is there polypeptide folding, but protein-protein interactions are also involved. For all proteins, but perhaps most important for aspherical ones, steric effects due to macromolecular crowding may modulate structure, stability, and folding. This is important as 5--40% of the available volume is occupied by various macromolecules in cells. To address these issues, I have used two model systems. Human mitochondrial co-chaperonin protein 10 (cpn10) is used for folding and assembly studies where the number of monomers is large (i.e., 7) and the fold of each monomer contains mostly beta-structure. In contrast, Borrelia burgdorferi VlsE is a football-shaped, monomeric protein with mostly alpha-helical structure that is employed in studies of how protein biophysical properties are affected by the surroundings in terms of membranes and crowding. Using in vitro biophysical and computational methods, my studies have identified the folding and assembly mechanism of cpn10: whereas heptamer unfolding precedes disassembly, a fraction of unfolded monomers assemble before folding while in the other fraction folding of monomers takes place before assembly. Furthermore, in crowded solutions, the helical structure of VlsE first increases and then, at more extreme conditions, a compact, non-native state with beta-sheet content can be populated that exposes an antigenic region. My results have implications for protein folding in general and for the function of these two proteins in particular ( i.e., chaperonin activity for cpn10 and Lyme disease for VlsE).