A grant has been awarded to Dr. Fraden at Brandeis University to develop microfluidic tools to crystallize proteins. Although the basic mechanism of protein crystallization is agreed upon by the research community, standard crystallization practices do not incorporate methods and instruments that optimally exploit the physical principles underlying crystallization. To overcome the deficiencies of current crystallization technologies Dr. Fraden will develop the Closed-Loop Protein Crystallizer (CLC), which incorporates the attributes of high-throughput, precision, and low volume that are characteristics of microfluidics. The conceptual underpinning of the CLC is to increase supersaturation until nanometer scale crystal nuclei are detected and then to dynamically vary the supersaturation conditions to control the rate of crystal growth.
The microfluidic protein crystallization device (CLC) consists of an array of wells to store hundreds of nanoliter volume drops containing protein solution. The device incorporates a semi-permeable membrane that allows the concentration of the protein solution to be varied, as well as a temperature stage that allows drops in separate wells to experience different temperatures. The CLC will incorporate Dynamic Light Scattering (DLS) which will be used to detect the onset of nucleation and subsequent crystal growth. The output of the DLS will be used in a closed-loop feedback mode to control the thermodynamic variables of concentration and temperature at the moment of crystallogenesis, which is the optimal moment to control crystallization.
The field of Structural Biology is dedicated to solving the structure of proteins motivated by the belief that ?structure implies function?. Structure ? function relationships are important for fundamental knowledge biomimetic engineering of new materials including new biomolecules. Protein structure is most commonly obtained through x-ray diffraction and crystallization is a necessary step in this process. One third of the 30,000 proteins that comprise the human genome reside in the membrane, yet only one human membrane protein has had its structure solved. Crystallization is the bottleneck in this process and the goal of this project is to eliminate this bottleneck by the development of the Closed Loop Crystallizer. Additionally, the cutting edge lab-on-a-chip instrumentation involved in this project will provide training to students in a technology sector vital to the US economy and scientific infrastructure.
Protein crystallization remains a bottleneck in the goal to determine the protein’s structure; essential for understanding protein function and for the rational design of pharmaceutics. The current paradigm governing crystallization is that crystallogenesis consists of a nucleation and growth process. However, this paradigm is largely untested and the most popular crystallization methods are not optimally designed to exploit the purported underlying mechanism. Several experimental constraints have hindered progress. (1) Proteins are precious, limiting the amount of protein per experiment. (2) The nucleation process is governed by random processes; therefore large numbers of identical experiments must be performed to obtain significant statistics. To address these two problems and to optimize crystallization technology we developed a microfluidic instrument specifically engineered for measuring and controlling the physically distinct processes of protein nucleation and growth while using the minimum amount of protein. We reduced sample volumes to less than 1 nl and performed experiments on 5000 samples simultaneously. Our results revealed that nucleation occurs via two processes; one as expected is consistent with direction nucleation, while a second process is consistent with a 2-step mechanism in which the proteins first aggregate and then transform into a crystal. The broader impact will be to improve the success rate of protein crystallization for structural biology which will advance fundamental understanding of protein function and the developement of pharmaceutics.
