Carbon Nanotube Atomic Force Microscopy for Proteomics and Biological Forensics

Abstract

The Human Genome Project was focused on mapping the complete genome. Yet, understanding the structure and function of the proteins expressed by the genome is the real end game. But there are approximately 100,000 proteins in the human body and the atomic structure has been determined for less than 1% of them. Given the current rate at which structures are being solved, it will take more than one hundred years to complete this task. The rate-limiting step in protein structure determination is the growth of high-quality single crystals for X-ray diffraction. Synthesis of the protein stock solution as well as X-ray diffraction and analysis can now often be done in a matter of weeks, but developing a recipe for crystallization can take years and, especially in the case of membrane proteins, is often completely unsuccessful. Consequently, techniques that can either help to elucidate the factors controlling macromolecular crystallization, increase the amount of structural information obtained from crystallized macromolecules or eliminate the need for crystallization altogether are of enormous importance. In addition, potential applications for those techniques extend well beyond the challenges of proteomics. The global spread of modern technology has brought with it an increasing threat from biological agents such as viruses. As a result, developing techniques for identifying and understanding the operation of such agents is becoming a major area of forensic research for DOE. Previous to this project, we have shown that we can use in situ atomic force microscopy (AFM) to image the surfaces of growing macromolecular crystals with molecular resolution (1-5) In addition to providing unprecedented information about macromolecular nucleation, growth and defect structure, these results allowed us to obtain low-resolution phase information for a number of macromolecules, providing structural information that was not obtainable from X-ray diffraction(3). For some virus systems, we have shown that AFM can already resolve some of the gross structural features of the virions themselves even in the absence of crystallization techniques (5). Our results show that the limitation on the applicability of this technique to structural studies of viruses and proteins is the size of the AFM tip which currently restricts the lateral resolution to about 10nm at best. The advent of carbon nanotube AFM probes made it possible to achieve unprecedented resolution in AFM imaging. Because single walled nanotubes have a radius of curvature of about 1nm, their use as AFM tips provides an order of magnitude improvement in lateral resolution. The purpose of this project was to use carbon nanotube tips to develop a new methodology for both determination of macromolecular structures and investigation of macromolecular crystallization. Our intent was to establish this methodology within the LLNL Directorates responsible for proteomics and biological forensics.