Our research program is comprised of closely allied applied and basic thrusts. The aim of our applied research is to harness the speed and specificity of biopolymer folding as a signal transduction mechanism in a novel and highly general class of real-time, “real-world” optical and electronic biosensors. In related studies we are exploiting the extraordinary cooperativity of folding for the creation of adaptive, responsive surfaces and materials. The goal of our basic research is the development and testing of a quantitative, first principles theory of the mechanism by which proteins fold. Related theoretical and experimental projects include intensive studies of the structure and dynamics of the unfolded state and the collective dynamics of the native state.

Folding-based sensors.

The ideal sensor will be sensitive, specific, versatile, small enough to hold in your hand, and selective enough to work even when faced with complex, contaminant-ridden samples. Given the affinity, specificity and generalizability of biomolecular recognition, biosensors have been widely touted for their potential to meet these challenging goals. To date, however, the translation of protein- and DNA-binding events into convenient, highly selective sensing platforms has proven problematic [51]. We have solved this problem by engineering binding-induced folding into proteins [54], peptides [66] and DNA [38, 53, 57, 58, 60, 62, 65] and employing the effect as a robust signal transduction mechanism.

 

Our folding-based sensors are rapid (minutes to seconds), sensitive (micromolar to femtomolar), fully electronic, and generalizable to an enormous range of protein, nucleic acid and small molecule targets. The sensors are also reagentless, greater than 99% reusable, and selective enough to be employed in (and re-used from) blood, soil and other grossly contaminated materials. Because of their sensitivity, background suppression, operational convenience and impressive scalability folding-based biosensors appear ideally suited for electronic, on-chip applications in pathogen detection, proteomics and genomics.

 

Principles of protein folding.

In an effort to understand why (more precisely, how) protein folding is some 80+ orders of magnitude more rapid than one would expect were it a random search of conformation space, we are conducting closely-coupled experimental and simulations-based studies of the folding process. Because we believe that the underlying physics of a problem is generally most readily apparent in the study of its simplest examples [21], the majority of our research efforts focus on the folding of simple, single-domain proteins. A primary goal of this research program is to address the question of why some simple, single domain proteins fold a million times more rapidly than others, and to use this vast rate dispersion to quantitatively test theories of the folding mechanism [reviewed in 42, 43, 44, 48]. To date we have demonstrated that the “folding energy landscape” of most simple, single domain proteins is extremely smooth; for example, even at the lowest temperatures we can probe their folding remains a highly cooperative process lacking any well populated, partially folded or misfolded intermediates [18, 22, 25, 44]. In the absence of complicating factors such as landscape roughness, intermediates and misfolding, what defines the relative folding rates of these simple proteins? Some years ago we made the empirical observation that the relative folding rates of single domain proteins can be accurately predicted using a very simple measure of the complexity of their native state topology [14, 21]. More recently we have put this empirical observation onto a firm theoretical foundation with the development of a simple, first principles model of protein folding kinetics, termed “the topomer search model” that accurately predicts the folding rates of single domain proteins [26, 32]. Ongoing research projects include several aimed at testing the detailed predictions produced by this promising new model [32, 33, 37, 43, 56].

In parallel with our studies of protein folding kinetics, we are increasingly focusing our efforts on studies of the unfolded state. These small angle X-ray scattering (SAXS), atomic force microscopy (AFM) and single molecule spectroscopy (SMS) studies all suggest that, contrary to a larger number of rather qualitative spectroscopic studies (reviewed in [52]), the denatured state populated at high levels of denaturant is exceedingly well described as a highly entropic [29], fully random coil [30, 45, 55] ensemble. Ongoing studies are focused on the somewhat more compact (relative to the random coil state) unfolded state populated in the absence of denaturant, and the relationship between this state and the random coil state [63,148].

 

Terahertz absorption spectroscopy and the collective dynamics of water and biomolecules.


Although the largest-scale harmonic vibrations of proteins (e.g., one domain beating against another) are often implicated in their function, to date these motions have proven exceedingly difficult to explore experimentally. Because these motions occur on the picosecond timescale, however, they can be probed using terahertz radiation (1 THz = 1/ps). Thus motivated, we have worked closely with the Allen group in physics to build the world’s highest precision, highest resolution absorbance spectrometer in this spectral regime. Using it, we have characterized the picosecond collective dynamics of proteins and their hydration shells [59,64, 133], the dynamics of water and other aqueous solutions [69, 54], and the potential of this technique as an astrobiological life-detection technology [40, 41].