Research - Protein crystallography

From molecules to crystals - structural studies of formyltetrahydrofolate synthetase

The bow inspector: This violin maker is inspecting the hair of a violin bow. Although bows are not necessary for producing sounds from a violin, their use provides a sustained sound distinct from the sound generated by plucking or striking the strings. To produce the sustained sound, the bow must contact the string with a stretched flexible material that has a specific set of "sticky" (tribological) properties. The contemporary approach to bows is to use stretched horse hair to provide the normal force and to cover the hair with rosin to provide the desirable tackiness. Because of the softness of the rosin, the structure and interaction between the rosin and bow hair is complicated. Without rosin, the horse hair is flaky, but with the application of rosin, the flakes are reduced, leaving a more homogeneous surface dotted with grains of rosin powder. Although the rosin powder appears similar to the protein crystals below, its chemical composition is primarily abietic acid.

Academic research at Wofford college

In addition to senior research, Wofford encourages the faculty to mentor students in academic research, particularly interdisciplinary efforts. During my introductory chemistry class, my chemistry professor, Dr. Ramin Radfar, approached me about a research opportunity for the fall of my junior year. As we discussed my previous work, I stated that I wanted to learn the techniques behind chemistry with a particular emphasis on the crystal fabrication. Dr. Radar gained funding from South Carolina Independent Colleges and Universities and oriented the experience towards the bio-chemistry: bacterial husbandry, protein harvesting, and crystal formation.

Motivation

Dr. Radfar's crystalographic studies of folates (specifically Formyltetrahydrofolate synthetase (FTHFS)) are motivated by finding new cancer therapies. Since the reproduction of cancer cells consumes energy at a rapid rate, understanding the structure of the folate enzymes that metabolize ATP can lead to cancer therapies that hinder the metabolization, thereby starving the cancer so it cannot reproduce as quickly. Of the twenty enzymes in folate biochemistry, three are cancer therapy targets.

My work with Dr. Radfar focused on optimizing the process of making crystals for use in the X-ray studies. For ease of access, we worked with FTHFS from a bacteria, and the final crystals were composed mostly of salt with the FTHFS enzymes encased inside. Fig 1 shows the structure of FTHFS identified in previous work by Dr. Radfar.

Method for forming crystals

Making the crystals started with gathering enough FTHFS to crystalize. I cultured moorella thermoacetica (a strain of e. coli), sonicated the cells (cell lysis), centrifuged the remains, extracted the desired protein, and confirmed the results with electrophoresis. After harvesting, I prepared the crystalizing wells for vapor diffusion. This technique shown in fig 2 relies on the fact that the saline+protein solution in the droplet will tend to evaporate, forcing the crystals to form as a precipitate. The size, number, and shape of the crystals depends upon the salt in the solution. We found that the largest cyrstals (and therefore most desirable crystals) formed with MgCl as the salt.

Experimental techniques used

Since the experience was geared towards learning the techniques involved in biochemistry, I was exposed to an array of equipment and processes including:

  • Autoclaving
  • Glassware preparation protocals
  • Titration
  • Vortex mixing
  • Bacterial growth
  • Encubating
  • Sonication
  • Centrifugation
  • Protein extraction
  • Electrophoresis
  • Vapor diffusion crystal growth