Fariba M. Assadi-Porter

Assistant scientist
University of Wisconsin-Madison
Postdoctoral training, University of Wisconsin-Madison
Ph.D. University of Wisconsin-Madison, Medical school

Biochemistry and NMR spectroscopy of brazzein sweet protein and the mechanism of its interaction with the sweet receptor

The central theme of my research is the application of nuclear magnetic resonance (NMR) spectroscopy to study structure function relationships in brazzein analogs and their interactions with the sweet receptor; using stable-isotope- assisted multinuclear NMR spectroscopy. The unique power of NMR lies in its ability to provide detailed chemical and structural information at an atomic level about molecules in solution--even when they are present in living cells or organisms. 

Brazzein is a naturally occurring sweet protein that was originally found in a tropical plant, Pentadiplandra brazzeana. Brazzein is only sweet to old world monkeys and human.  Brazzein is a new generation of low-calorie sweeteners with functional health attributes that does not involve carbohydrates. The availability of foods low in sugar content yet high in flavor is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes.

My initial focus has been on developing expression system for the production of this difficult to make naturally sweet protein. We synthesized a gene coding for brazzein and developed an efficient bacterial production system. The fusion protein constructs are expressed either as an insoluble product, which we solubilize and fold, or as soluble form. The conditions we developed for folding and oxidation of the disulfides and cleavage of the fusion protein lead to a product with native structure (as determined by NMR spectroscopy) and with full activity as a sweetener (as determined by taste tests).  This methodology, along with quick-change mutagenesis, has allowed us to make a variety of brazzein mutants. We discovered mutants with sweet-taste properties that appear to be superior to those of the wild-type protein. The presence of positive charges on the surface of brazzein enhances sweetness; however, mutating the charges causes sweetness to decline. Our current model suggests a ?multi-site? binding mechanism for brazzein to the sweet receptor. We are currently investigating the molecular mechanism of protein sweetener interaction with the human sweet receptor by NMR spectroscopy.

My recent endeavors include developing methods to determine early onset of disease using a multi-displinary approach.  One central question is whether or not an animal is suffering from catabolism due to disease onset? To answer this question, we use new innovative method, using isotope changes in breath to distinguish for example bacterial infections from viral infections in humans and other animals.

We plan to systematically enhance our current metabolic analysis methodology to rapidly identify early and reliable biomarkers of diseases with seemingly unrelated complex phenotypes using polycystic ovary syndrome (PCOS) as our case model.