Design and characterization of a protein fold switching network.

To better understand how amino acid sequence encodes protein structure, we engineered mutational pathways that connect three common folds (3α, ß-grasp, and α/ß-plait). The structures of proteins at high sequence-identity intersections in the pathways (nodes) were determined using NMR spectroscopy and analyzed for stability and function. To generate nodes, the amino acid sequence encoding a smaller fold is embedded in the structure of an ~50% larger fold and a new sequence compatible with two sets of native interactions is designed. This generates protein pairs with a 3α or ß-grasp fold in the smaller form but an α/ß-plait fold in the larger form. Further, embedding smaller antagonistic folds creates critical states in the larger folds such that single amino acid substitutions can switch both their fold and function. The results help explain the underlying ambiguity in the protein folding code and show that new protein structures can evolve via abrupt fold switching.

Engineering subtilisin proteases that specifically degrade active RAS.

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Engineering Photosystem I Complexes with Metal Oxide Binding Peptides for Bioelectronic Applications.

Conventional dye-sensitized solar cells comprise semiconducting anodes sensitized with complex synthetic organometallic dyes, a platinum counter electrode, and a liquid electrolyte. This work focuses on replacing synthetic dyes with a naturally occurring biological pigment-protein complex known as Photosystem I (PSI). Specifically, ZnO binding peptides (ZOBiP)-fused PSI subunits (ZOBiP-PsaD and ZOBiP-PsaE) and TiO2 binding peptides (TOBiP)-fused ferredoxin (TOBiP-Fd) have been produced recombinantly from Escherichia coli. The MOBiP-fused peptides have been characterized via western blotting, circular dichroism, MALDI-TOF, and cyclic voltammetry. ZOBiP-PSI subunits have been used to replace wild-type PsaD and PsaE, and TOBiP-Fd has been chemically cross-linked to the stromal hump of PSI. These MOBiP peptides and MOBiP-PSI complexes have been produced and incubated with various metal oxide nanoparticles, showing increased binding when compared to that of wild-type PSI complexes.

Photoelectrochemistry of photosystem I bound in nafion.

Developing a solid state Photosystem I (PSI) modified electrode is attractive for photoelectrochemical applications because of the quantum yield of PSI, which approaches unity in the visible spectrum. Electrodes are constructed using a Nafion film to encapsulate PSI as well as the hole-scavenging redox mediator Os(bpy)2Cl2. The photoactive electrodes generate photocurrents of 4 µA/cm(2) when illuminated with 1.4 mW/cm(2) of 676 nm band-pass filtered light. Methyl viologen (MV(2+)) is present in the electrolyte to scavenge photoelectrons from PSI in the Nafion film and transport charges to the counter electrode. Because MV(2+) is positively charged in both reduced and oxidized states, it is able to diffuse through the cation permeable channels of Nafion. Photocurrent is produced when the working electrode is set to voltages negative of the Os(3+)/Os(2+) redox potential. Charge transfer through the Nafion film and photohole scavenging at the PSI luminal surface by Os(bpy)2Cl2 depends on the reduction of Os redox centers to Os(2+) via hole scavenging from PSI. The optimal film densities of Nafion (10 µg/cm(2) Nafion) and PSI (100 µg/cm(2) PSI) are determined to provide the highest photocurrents. These optimal film densities force films to be thin to allow the majority of PSI to have productive electrical contact with the backing electrode.

Molecular interactions between photosystem I and ferredoxin: an integrated energy frustration and experimental model.

The stromal domain (PsaC, PsaD, and PsaE) of photosystem I (PSI) reduces transiently bound ferredoxin (Fd) or flavodoxin. Experimental structures exist for all of these protein partners individually, but no experimental structure of the PSI/Fd or PSI/flavodoxin complexes is presently available. Molecular models of Fd docked onto the stromal domain of the cyanobacterial PSI site are constructed here utilizing X-ray and NMR structures of PSI and Fd, respectively. Predictions of potential protein-protein interaction regions are based on experimental site-directed mutagenesis and cross-linking studies to guide rigid body docking calculations of Fd into PSI, complemented by energy landscape theory to bring together regions of high energetic frustration on each of the interacting proteins. The results identify two regions of high localized frustration on the surface of Fd that contain negatively charged Asp and Glu residues. This study predicts that these regions interact predominantly with regions of high localized frustration on the PsaC, PsaD, and PsaE chains of PSI, which include several residues predicted by previous experimental studies.

Structure and function of POTRA domains of Omp85/TPS superfamily.

The Omp85/TPS (outer-membrane protein of 85 kDa/two-partner secretion) superfamily is a ubiquitous and major class of ß-barrel proteins. This superfamily is restricted to the outer membranes of gram-negative bacteria, mitochondria, and chloroplasts. The common architecture, with an N-terminus consisting of repeats of soluble polypeptide-transport-associated (POTRA) domains and a C-terminal ß-barrel pore is highly conserved. The structures of multiple POTRA domains and one full-length TPS protein have been solved, yet discovering roles of individual POTRA domains has been difficult. This review focuses on similarities and differences between POTRA structures, emphasizing POTRA domains in autotrophic organisms including plants and cyanobacteria. Unique roles, specific for certain POTRA domains, are examined in the context of POTRA location with respect to their attachment to the ß-barrel pore, and their degree of biological dispensability. Finally, because many POTRA domains may have the ability to interact with thousands of partner proteins, possible modes of these interactions are also explored.

Crystal structure of the anthrax drug target, Bacillus anthracis dihydrofolate reductase.

Spores of Bacillus anthracis are the infectious agent of anthrax. Current antibiotic treatments are limited due to resistance and patient age restrictions; thus, additional targets for therapeutic intervention are needed. One possible candidate is dihydrofolate reductase (DHFR), a biosynthetic enzyme necessary for anthrax pathogenicity. We determined the crystal structure of DHFR from B. anthracis (baDHFR) in complex with methotrexate (MTX; 1) at 2.4 Angstrom resolution. The structure reveals the crucial interactions required for MTX binding and a putative molecular basis for how baDHFR has natural resistance to trimethoprim (TMP; 2). The structure also allows insights for designing selective baDHFR inhibitors that will have weak affinities for the human enzyme. Additionally, we have found that 5-nitro-6-methylamino-isocytosine (MANIC; 3), which inhibits another B. anthracis folate synthesis enzyme, dihydropteroate synthase (DHPS), can also inhibit baDHFR. This provides a starting point for designing multi-target inhibitors that are less likely to induce drug resistance.