Nature has provided many proteins with catalytic, structural, or physical properties that render them useful as tools in chemical, biochemical, or biotechnological applications. However, the evolutionary forces that have generated this myriad of proteins are very different from the quite unnatural demands humans place on their tools. It is often necessary to "improve" natural proteins to make them better tools. For example, to be useful for industrial applications an enzyme may have to be engineered to be stable and active at high temperature or in the presence of solvents that a protein would not encounter in Nature. As difficult as it may be to generate ideal 'protein-based' tools, the effort is justified because proteins can have such remarkable structural (e.g. spider silk), catalytic (enzymes), or physical (e.g. green fluorescent protein) properties. It is for this reason that Protein Engineering, i.e. the directed evolution of existing proteins or the creation of de novo proteins with no structural or functional homologue in Nature, is a critical direction of research.
My specific focus is on the use of Protein Engineering for the development of genetically encoded fluorescent labels and reporters for imaging and manipulation of biochemistry in living cells. Fluorescent proteins (FPs), such as the Aequorea jellyfish green fluorescent protein (GFP) (see Figure 1), are nearly ideal fluorescent labels because they can be expressed in a variety of different organisms and fused to many different proteins of interest with little or no effect on either proteins function. Reporters based on fluorescence resonance energy transfer (FRET) between two engineered variants of GFP, a cyan FP (CFP) and a yellow FP (YFP), have found great utility in cell biology (Figure 2A). An ideal complement to the numerous reporters of this type would be a spectrally distinct red-shifted FRET pair that would allow simultaneous imaging of two reporters to determine causal relationships between biochemical processes. Efforts to develop such a FRET pair, using both red-shifted FPs and in situ labeling strategies, will be a major focus of my research. An alternative reporter design is to engineer FP variants in which binding of a small molecule directly modulates the fluorescence spectrum (Figure 2B). This type of sensor has been reported but current designs lack generality. To overcome this limitation I plan to employ Protein Engineering to create de novo sensors that could be tailored to detect any small molecule of interest.
While the chemical composition of proteins is rather simple (a linear polypeptide composed of the 20 common amino acids) their 3-dimensional structures are fantastically complex. However, moderate modifications of complex protein structures such as point mutations, insertions and deletions, or fusion to another protein, are simple procedures that can be achieved in just a few hours of work using the techniques of molecular biology. The challenge of Protein Engineering is to use these simple molecular biology techniques to produce a new protein that is either an improvement over the original or has acquired some completely new function. Generally speaking, we have not yet reached the point where we can predict what effect a given point mutation will have on a given protein's structure and function. For this reason, the most effective approach to Protein Engineering is to rely on high throughput screening of libraries of mutated proteins with selection of those rare variants that are improved over the parent protein. Methods of screening can range from very low throughput (manual assay of individual clones), to medium throughput (96-well plate format or screening of bacterial colonies), to very high throughput (phage display and fluorescence activated cell sorting (FACS)). Research in my laboratory will be highly multidisciplinary with projects that involve organic synthesis, molecular biology, protein crystallography, molecular modeling, and live cell fluorescence microscopy. I anticipate that in the course of their graduate research a typical student will obtain a thorough training in at least two of the above disciplines, will be very familiar with the others, and will have a built a strong foundation on which to start a successful and rewarding career in chemistry, biochemistry, or biotechnology.