Researchers Give Green Light To Protein Folding

In the July issue of Nature Biotechnology magazine, Geoffrey Waldo and his colleagues describe their method for Rapid Protein Folding Assay (RPFA). The process, which uses readily available assay materials and techniques, allows for the rapid analyses of large numbers of proteins and gives a visible test result proportionate to the percentage of soluble protein in the sample.

According to Waldo, "the principal application for our protein folding assay is the rapid identification of soluble proteins. This assay will be particularly useful to medical researchers doing drug development and also to scientists working in the emerging field of proteomics -- the study of the structures and functions of all the proteins encoded by the genome."

As the basic building blocks of cells, soluble proteins act as catalysts and controllers for numerous chemical reactions, in addition to helping give cells, organs and tissues their shape and structure. Individual protein molecules can assemble themselves in a variety of different three-dimensional structures with intricate origami-like folds. The range of shapes is governed by interactions among the atoms that make up the protein and the folding process influences functionality. If the protein folds correctly during formation, it will function correctly as a soluble protein. Misfolded proteins are usually insoluble and useless to a cell.

"We believe," said Waldo, "that some currently incurable diseases, such as Alzheimer's and Huntington's, are associated with protein misfolding. Since our assay allows researchers to rapidly check proteins for misfolding, this should speed up the development of new assays and drugs to prevent or reverse protein misfolding. Of course, it's not as simple as it sounds, but it's certainly a step in the right direction."

The RPFA method works by linking or fusing the DNA of the protein being assayed to the DNA for green fluorescent proteins (GFP). The hybrid protein created by this linking has the characteristics of both the GFP and the protein being assayed. If the protein being produced, or expressed, folds correctly then the attached GFP will also fold correctly as it is expressed. If the protein being expressed does not fold correctly, then the GFP will not fold correctly. Because correctly folded GFP fluoresces, or glows, green when exposed to blue or ultraviolet light the presence of the green glow in a sample is an indicator that the protein being assayed is not misfolded and therefore likely soluble.

"An important attribute of RPFA," said Waldo, "is its ability to separate protein function from protein folding. Since the assay only measures the successful folding of a protein, it neatly bypasses the need to know a protein's function. This allows the analysis of proteins whose function might be difficult, or even impossible, to measure. This ability gives RPFA universality."

Universality is just what an assay needs to meet the challenges facing proteomics. With about 80,000 human genes now identified by the Human Genome Project, the task ahead is to try to understand the structure and function of all the proteins encoded by those genes. The goal is a better understanding of the way proteins carry out their work, the relationship of proteins to different diseases and the mechanisms by which therapeutic proteins, such as insulin, prevent or counter disease. This enormous research effort requires tools like RAPF to accomplish its goal.

The method joins a battery of techniques being developed at Los Alamos to obtain structures of the proteins encoded by the human genome. SOLVE, a computer program developed by Los Alamos researchers Tom Terwilliger and Joel Berendzen, lets scientists get three-dimensional maps of proteins from x-ray pictures of protein crystals with unprecedented speed and simplicity. RPFA provides the needed soluble protein for growing the protein crystals.

Waldo stresses the merits of the assay. "Not only is the Rapid Protein Folding Assay relatively quick and accurate, but because it uses the folding of GFP to report on the folding of the test protein, it is a self-contained technology. It eliminates the time-consuming, one-at-a-time development of functional assays that are reliant on external reagents. That saves laboratories both time and money."

Los Alamos National Laboratory is operated by the University of California for the Department of Energy.