| Biomolecular engineering is a rapidly expanding field that integrates disciplines, such as chemistry and biology, to address a number of different scientific problems. More specifically, the use of biomolecular engineering techniques can contribute to the areas of protein and biosensor design. Here, biomolecular engineering tools were utilized for two important reasons: to evaluate how geometry plays a role in the formation of crosslink protein derived cofactors (CPDCs) and to generate biosensors to detect biomolecules using aptamers as the selective recognition element.;The analysis of the geometric constraints involved in CPDC formation was achieved by using the SiteSearch algorithm of the Dezymer biomolecular modeling package. As a result of this evaluation it was observed that four CPDCs had significantly higher formation propensities than the common disulfide bond, which was run as a control. Also, a large number of CPDC amino acid pairs with short sequence separations ( i, i + (1-5)) were reported. Identifying these near-sequence crosslinked cofactors could be problematic using the current proteolysis/mass spectrometric techniques. One of the proteins from the 500 protein library, rat intestinal fatty acid binding protein (IFABP), was selected for experimental evaluation and the reported sites were constructed using recombinant DNA technology. Using photooxidative methods, geometrically predicted YC crosslink formation was evaluated using fluorescence and other analytical techniques.;A second project involves the extension of a modular protein-based quantum dot biosensor to an aptamer-based sensor for thrombin detection. The previously reported prototype system used a redox active ruthenium(II) complex, semiconducting nanoparticles and maltose binding protein (MBP) to detect the sugar maltose. The aptamer-based system developed here uses the well characterized thrombin binding aptamer as the selective recognition element to detect the serine protease thrombin. The thrombin biosensor was demonstrated to be both selective (when challenged with other proteins) and sensitive (responding to thrombin concentrations as low as 500 pM). A response to thrombin was also shown using semiconducting nanoparticles that emit and absorb in the red allowing for the potential use of this sensor in complex solutions such as blood. |
