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Protein folding studies give insight into the mechanism and environmental conditions as to the most optimal environment for active proteins. Under physiological conditions, proteins typically exist in their non-active unfolded structure. Upon binding of a catalyst, folding into the proteins' active conformation is initiated followed by an unfolding event that occurs with the loss of the catalyst in the environment. To monitor the process of the folding event, a change in the optical properties of the proteins must occur for detection by spectroscopic means. Proteins, while exhibiting change of secondary and tertiary structure as seen through circular dichroism and UV absorption, can not be mechanistically studied with current technology. We can not confirm the exact confirmation changes that take place during binding. The ability to detect binding events in biological materials has been limited mainly to the use of radioactive ligand labeling assayed by either autoradiography or through conjugation with biotin or other flourochromes. While both methods of analysis give accurate results, they are usually inconvenient to work with due to the radioactive elements and are time consuming due to exposure times of up to several weeks. Binding events as detected through optical spectroscopy are also limited with the use of conventional flourophores. Flourophores can typically only absorb light at a very specific wavelength making it difficult to excite the electrons in the system. The then emit light in a very broad range. In a system with two flourophores, it becomes difficult to distinguish between the two separate emissions due to cross-talking of the peaks. A more efficient system would be one that exhibited the exact opposite of the common flourophores. This would be one that can absorb light at almost any wavelength and then only emits light at a very specific wavelength. This type of system could be accomplished with the use of nanocrystals. Particles in the range of 2nm in diameter up to almost 100 nm exhibit physical and optical properties that are not seen in bulk material. This is due to quantum confinement effects. At this size, quantum mechanics plays a large role in the optical properties of these materials. Semiconducting CdSe nanocrystals and metallic Au nanocrystals are the most commonly studied nanocrystals. Their ability to have a narrow, tunable, symmetric emission spectrum allow them to be in some cases superior to the existing flourophores. To examine the use of nanocrystals in bio-material based sensors, it is first necessary to understand the binding properties that would be needed in such a system. In this report, we present a method of binding nanocrystals to specific sites on a peptide for controlled assembly through stoichiometric addition. Both CdSe and Au nanocrystals were studied due to their accessibility. For binding with biomaterials, the organically soluble nanocrystals must first be modified to be water soluble because the peptide is also water soluble. A methods for the ligand exchange of CdSe is also presented here to change the surface properties of the nanocrystals. Upon success of appending nanocrystals to a short peptide, electron and energy transfer studies can be done to determine the efficiency of this system. Further studies of larger proteins and more specific binding would also have to be done to eventually create a usable biosensor. Return to the RISE 2000 project list |
