Biological macromolecules are the machinery of life and understanding their function helps scientists to develop new drug treatments that target specific human diseases. In this regard, crystallization is routinely employed for the understanding of the molecular structures and the interactions of proteins with other biological and non-biological materials. Despite the existence of a plethora of crystallization techniques, there is still a need for a technique that affords for better control over the crystallization procss, in terms of producing high-quality crystals of peptides and proteins in a significantly shorter tim scale. In this STTR Phase I proposal, we will construct a prototype crystallization instrument based on the metal-assisted and microwave-assisted evaporative crystallization (MA-MAEC) technique for the rapid crystallization of peptides and proteins in minutes or hours (total crystallization time), which typically can take up to several weeks to complete using conventional crystallization techniques. In this regard, we have chosen Amyloid precursor protein (APP) and its components as biological macromolecules of interest. APP is most commonly studied as the precursor molecule whose proteolysis generates beta amyloid (A42) peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.
Alzheimer's disease is an age-related, non-reversible brain disorder that develops over a period of years and the sixth leading cause of death in USA. Recent studies estimates that there are 5.2 million Americans of all ages have this disease. In addition, the number of people age 65 and older with Alzheimer's disease is estimated to reach 7.1 million by 2025. There are three major markers in the brain that are associated with the process of Alzheimer's disease: Amyloid plaques, neurofibrillary tangles and loss of connections between neurons. Amyloid precursor protein (APP), which plays an important role in the development of Alzheimer's disease, is a membrane protein with several significant domains (growth factor domain (GFD), copper binding domain (CuBD), Kunitz-type protease inhibitor domain. Most importantly, the proteolysis of APP generates a neurotoxic A peptide, which can affect neural functions and trigger cell death. In addition, A peptide can aggregate into small soluble oligomers, eventually leading to the amyloid plaques observed in brains of patients who had Alzheimer's disease. Although the complete crystal structure of APP has not been solved, most of its individual domains (except A) have been crystallized. In this regard, we propose new instrumentation (i.e, named iCrystal) and crystallization platforms that allows for rapid crystallization of APP and it components. We envision that the iCrystal system can be applied to the crystallization of any biological macromolecule related to human diseases.
