Extremophilic chaperones are promising because of their restorative functions within the cell, their ability to facilitate protein folding under harsh conditions, and their overall minimal protein folding systems (1). An often-overlooked aspect of chaperones is their exquisite quaternary structures, with different proteins forming ring structures (chaperonins), clamps (HSP70, prefoldin), spheres (sHSP), and filaments (chaperonins, γ prefoldin). In this presentation I describe use of chaperones for engineering of stress pathways in mesophilic hosts and exploitation of chaperones to build tunable nanostructures.

We have investigated the effects of heterologous expression of hyperthermophilic molecular chaperones on the survival phenotypes of E. coli TUNER strain. We first demonstrate that individual heterologous expression of three different molecular chaperones (prefoldin, small heat shock protein, or thermosome) can increase the temperature tolerance of E. coli. We demonstrate that chaperone function of these proteins as responsible for the survival phenotype in E. coli. We extend this approach to test for increased survivability against the biofuels ethanol and butanol. We conclude by testing additivity effects of alternative protein-stabilizing solutes (e.g., trehalose) with these hyperthermophilic chaperones.

Protein self-assembly is a particularly attractive option for bottom-up creation of tunable smart materials and hybrid inorganic/organic functional devices. Proteins can potentially serve as scaffolds for a host of nanostructured materials by self-assembling them at precise spatial distances and directing their placement onto solid supports at defined locations. We wish to find a general approach to build protein assemblages of arbitrary geometry over multiple length scales. To achieve this goal, the following benchmarks must be overcome: (A) a suitable protein template must be identified. (B) Control of the length of the protein template must be demonstrated. (C) Genetic modification of the template must be established. (D) Absolute control over a single dimension (e.g., length) must be engineered. These initial four steps directly lead to creation of more complex shapes and geometries. This presentation elucidates the progress we have made in these areas.

We have recently discovered a new type of filamentous protein (the γ prefoldin, or γ-PFD) from the hyperthermophile Methanocaldococcus jannaschii (2, 3). The filaments are polydisperse in length (microns) and monodisperse in width (8.5 nm) and height (4 nm). They are also remarkably stable, maintaining their structure up to at least 100ºC and in 7 M guanidinium chloride. With the overall aim of utilizing γ-PFD filaments as structural components in 3-D nano-scale architectures, we describe here the kinetic scheme behind γ-PFD filament formation and demonstrate control over filament length by rational design of an extension resistance mutant protein (so-called TERMs, or thermophilic extension resistant mutants) (4). We then demonstrate self-assembly of this filament with functionalized ends and a different functionalized interior. The next phase of this project involves using powerful genetic selection methodology to create periodic 3-D architectures of arbitrary geometry, and progress toward this end will be discussed.

1. Laksanalamai, P., Whitehead, T. A. & Robb, F. T. (2004) Nat Rev Microbiol 2, 315-24. 2. Boonyaratanakornkit, B. B., Simpson, A. J., Whitehead, T. A., Fraser, C. M., El-Sayed, N. M. & Clark, D. S. (2005) Environ Microbiol 7, 789-97. 3. Whitehead, T. A., Boonyaratanakornkit, B. B., Hollrigl, V. & Clark, D. S. (2007) Protein Science 16, 626-634. 4. Whitehead, T.A, Meadows, A.L., Clark, D.S., submitted.