Browsing by Author "Hussain, Faiza"

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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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    Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR Family Chimeras
    (American Chemical Society, 2014) Shis, David L.; Hussain, Faiza; Meinhardt, Sarah; Swint-Kruse, Liskin; Bennett, Matthew R.
    In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits.
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    Sources of Variability in a Synthetic Gene Oscillator
    (Public Library of Science, 2015) Veliz-Cuba, Alan; Hirning, Andrew J.; Atanas, Adam A.; Hussain, Faiza; Vancia, Flavia; Josić, Krešimir; Bennett, Matthew R.
    Synthetic gene oscillators are small, engineered genetic circuits that produce periodic variations in target protein expression. Like other gene circuits, synthetic gene oscillators are noisy and exhibit fluctuations in amplitude and period. Understanding the origins of such variability is key to building predictive models that can guide the rational design of synthetic circuits. Here, we developed a method for determining the impact of different sources of noise in genetic oscillators by measuring the variability in oscillation amplitude and correlations between sister cells. We first used a combination of microfluidic devices and time-lapse fluorescence microscopy to track oscillations in cell lineages across many generations. We found that oscillation amplitude exhibited high cell-to-cell variability, while sister cells remained strongly correlated for many minutes after cell division. To understand how such variability arises, we constructed a computational model that identified the impact of various noise sources across the lineage of an initial cell. When each source of noise was appropriately tuned the model reproduced the experimentally observed amplitude variability and correlations, and accurately predicted outcomes under novel experimental conditions. Our combination of computational modeling and time-lapse data analysis provides a general way to examine the sources of variability in dynamic gene circuits.