Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNA and pol δ. During this process, the interactions of RPA, RFC and pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNA and pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPA•DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPA•DNA interactions in RPA-dependent DNA metabolic processes.

Interplay of macromolecular interactions during assembly of human DNA polymerase δ holoenzymes and initiation of DNA synthesis.

In humans, DNA polymerase δ (Pol δ) holoenzymes, comprised of Pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand DNA replication, initiation of leading strand DNA replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a coordinated process involving the major single strand DNA-binding protein complex, replication protein A (RPA), the processivity sliding clamp loader, replication factor C (RFC), PCNA, and Pol δ. Each of these factors interact uniquely with a P/T junction and most directly engage one another. Currently, the interplay between these macromolecular interactions is largely unknown. In the present study, novel Förster Resonance Energy Transfer (FRET) assays reveal that dynamic interactions of RPA with a P/T junction during assembly of a Pol δ holoenzyme and initiation of DNA synthesis maintain RPA at a P/T junction and accommodate RFC, PCNA, and Pol δ, maximizing the efficiency of each process. Collectively, these studies significantly advance our understanding of human DNA replication and DNA repair.

Covalent and non-covalent strategies for the immobilization of Tobacco Etch Virus protease (TEVp) on superparamagnetic nanoparticles.

Proteases with highly specific activities have numerous applications, including the cleavage of affinity tags (Flag; HA; His6X) and solubility promoting partners (GST; MBP) within the context of protein isolation and purification schemes. However, commercially sourced proteases such as Tobacco Etch Virus protease (TEVp) and Human Rhinovirus (HRV) 3C protease are typically applied as single use aliquots, which limits their cost-effectiveness. In addition, the presence of residual proteases in downstream applications can complicate analysis of the protein of interest. Thus, the creation of immobilized, reusable site-specific proteases would be of significant value to the life science community. In this work, we explore two strategies for the immobilization of TEV protease onto superparamagnetic iron oxide nanoparticles (SPIONs). In one strategy, a MBP-TEVp-Streptavidin fusion protein is immobilized on biotin-functionalized SPIONs. In a second strategy, TEV protease is covalently coupled onto SPIONs directly, via amine-mediated attachment, and indirectly, via HALO-tag mediated attachment. We demonstrate activity of our immobilized proteases in the presence of a MBP-GFP fusion protein containing the TEV protease target sequence (ENLYFQ|S). We then analyze time-dependent activity, longevity, and reuse of these immobilized protein preparations, comparing each approach. The protease immobilization strategies described in this work may be useful tools for simplifying challenging protein purification protocols, in addition to providing general methods for enzyme immobilization on SPIONs.

Impact of Local Electrostatics on the Redox Properties of Tryptophan Radicals in Azurin: Implications for Redox-Active Tryptophans in Proton-Coupled Electron Transfer.

Tyrosine and tryptophan play critical roles in facilitating proton-coupled electron transfer (PCET) processes essential to life. The local protein environment is anticipated to modulate the thermodynamics of amino acid radicals to achieve controlled, unidirectional PCET. Herein, square-wave voltammetry was employed to investigate the electrostatic effects on the redox properties of tryptophan in two variants of the protein azurin. Each variant contains a single redox-active tryptophan, W48 or W108, in a unique and buried protein environment. These tryptophan residues exhibit reversible square-wave voltammograms. A Pourbaix plot, representing the reduction potentials versus pH, is presented for the non-H-bonded W48, which has potentials comparable to those of tryptophan in solution. The reduction potentials of W108 are seen to be increased by more than 100 mV across the same pH range. Molecular dynamics shows that, despite its buried indole ring, the N-H of W108 hydrogen bonds with a water cluster, while W48 is completely excluded from interactions with water or polar groups. These redox properties provide insight into the role of the protein in tuning the reactivity of tryptophan radicals, a requirement for controlled biological PCET.

protaTETHER - a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc-GFP fusion protein.

Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemed protaTETHER, utilizes reiterative oligomer design, PCR-mediated linker library generation, and restriction enzyme-free cloning methods in a straightforward, three-step cloning process. We utilize a fusion between the catalytic subunit of cAMP-dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHER method, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc-GFP fusions, and use these results to select a PKAc-GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression.