Secondary Protein Structure And Prion Formation Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Neurodegenerative diseases are an on-going topic of exploration in the scientific and medical communities. One class of such diseases is caused by the infectious isoform of cellular protein prions. Diseases formed by compromised prions are found throughout various species. Scarpie in sheep, bovine spongiform encephalopathy in cows, and Creutzfeldt - Jakob disease in humans are the common points of prion disease exploration. This experiment examines the idea that a conformational change from α-helices to β-sheets in cellular prion proteins is responsible for the creation of the isoform accountable for the neurodegenerative disease. Prior to experimentation, it was determined that the point at which the normal prion becomes an infectious one is post-translational (post protein synthesis). As a result, the experiment identifies chemical modification as another probable cause for the prion change. However, there is little evidence to support the chemical modification claim, and experimental efforts were geared toward the support of the conformational change theory.

The experiment was conducted using the prion isoform found in scarpie (PrPsc), and its derivative PrP 27-30. For prion propagation, the brains of Syrian gold hamsters were extracted after death by CO2 asphyxiation (suffocation). The brains were immediately frozen in liquid nitrogen, and stored at -80°C until needed. Observations of both the scarpie isoform, and normal cellular prions (PrPc) were needed. The secondary structure of both prion types were observed using Fourier transform methods (FTIR). Fourier transform spectroscopy utilizes electromagnetic or other forms of radiation to give an optical view of the molecular structure of a species. Being that conservation of the protein structures was important to experimental results, both the cellular and isoform prions were extracted using non-degenerative (non-abrasive) methods. The normal cellular prion was prepared from a hundred normal hamster brains. Test fractions were prepared using Zwittergent 3-12 and centrifuging techniques. The scarpie isoform, and its derivative, PrP 27-30, were extracted and purified from scarpie-infected Syrian hamster brains. All prion types were then denatured by boiling in sample buffer.

Analysis was carried out with the use of SDS-PAGE; a technique is used for the separation of DNA, RNA, or protein molecules, by application of an electric field to a gel matrix. The newly resolved proteins were then stained with a silver solution to enhance observation. Along with Fourier transform methods, Immunogold labeling was a determinant selected for prion identification.

The variables of concern in this experiment were the structural elements of the three prion types; PrPc (normal/ control), PrPSC , and PrP27-30. Under spectroscopy, the PrPc prion showed characteristics that inferred the presence of a high α-helical content. This was realized by the presence of the 'amide I' bond. Both prion isoforms (PrPSC, and PrP27-30) were determined to have high β-sheet content based on their spectra. Based on deconvolutions found throughout individual spectrum, numerical estimates of the α-helix/β-sheet content of each prion type were made. The PrPc was found to consist of 42% α-helical content and 3% β-sheet makeup. The PrPSC and PrP 27-30 spectra revealed higher β-sheet contents; 43% and 54% respectively. For support, these results were compared to pre-experimental predictions derived from a neural network algorithm. In both cases, the idea that a conformational change was associated with the shift from PrPc to PrPSC was supported. However, the values recorded by both techniques (FTIR, network algorithm) were different.

With respect to PrPc, additional insight was obtained by CD spectra analysis. This technique was used based on its ability to detect high α-helical content. The α-helical content of 36% obtained was deemed consistent with the discussed results. Under Electron microscopy (uses a high power microscope), both the normal and isoform (PrPSC) versions of the prion appeared as aggregates when dried. The PrP27-30 derivative gave a rod-like appearance. These rod-like formations were determined to be amyloidal polymers of PrP27-30. Being that the intermolecular forces associated with β-sheets, of which a high concentration exists in the PrP27-30 prion, is associated with polymer formation; it supports the idea that secondary structure is linked to infectious prion formation.

Arguably, the results obtained in this experiment offered supporting evidence for the link between high β-sheet content and infectious prion formation. For example, under electron microscopy, experimenters were able view amyloidal forms found in species suffering from the degenerative disease. However, the experiment failed to illustrate whether or not a conformational change occurs, or is plausible. The tests carried out used a prion in which experimenters induced a conformational shift (PrP27-30). Although experimentally, shifts in the concentrations of α-helical and β-sheet structures show probable cause for the prion isoform formation these results do not illustrate how or if the conformational shifts occur.

Notably, the research carried out is of value to neurodegenerative research. The more insight that is given into such a problem can only aid in the establishment of its solution. The idea of a conformational change as the responsible party in the formation of the prion isoform is notable input. However, the inferences made in this experiment are not enough to establish it as a valid theory. Research into if and how these conformational changes occur, would be a superior contribution to the research community.