Literature Review Regarding Prion Protein Experiment Paper Biology Essay

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Pan, K. M., M. Baldwin, J. Nguyen, M. Gasset, A. Serban, D. Groth, I. Mehlhorn, Z. Huang, R. Fletterick, F. Cohen. "Conversion of Alpha-helices into Beta-sheets Features in the Formation of the Scrapie Prion Proteins." PNAS 90.23 (1993): 10962-0966. Print


In their paper, "Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins," Pan et al from the University of California, San Francisco, used nondenturing methods to purify and characterize several forms of the prion protein in an effort to uncover the folding nature of the prion proteins and how the abnormal isoform of the protein develops in the body in living organisms. Up until then, it was known that the abnormal isoform of the scrapie protein existed due to a post-translational process that occurs in endosomes, which are compartments of cells that directs molecules from the plasma membrane to the lysosome. Using denaturing agents such as sodium dodecyl sulfate (SDS) and guanidine hydrochloride, Pan et al are able to purify the normal as well as abnormal forms of the prion protein from Syrian golden hamsters and can then study the secondary structure of the extracted and purified proteins using Fourier-transform infrared (FTIR) spectroscopy, circular dichroism (CD) studies, as well as visualization of the proteins fibers using electron microscopy (EM). It was found that the in situ conformational change from the normal prion protein (42% alpha helix content) to the abnormal scrapie protein (43% beta-sheet content) is responsible for the propagation of the prion disease. Although significant work was done by Pan et al to lead to such a finding, there is still much debate on the topic of prions as the exact mechanism of the conformational change is not determined by these studies, as well as the cause of such a transforming event yet remain unknown. Thus, although this paper shows experimental data that confirms previous hypotheses, it is lacking an in-depth analysis of the nature of the prion proteins.


As described above, Pan et al performed many experiments to purify and characterize the prion protein (PrPC), the abnormal isoform of the prion protein that cause neurodegenerative diseases (PrPSc), as well as the protease-resistant core of (PrPSc), called PrP 27-30. Because denaturing conditions are avoided, low pH, urea concentration, and the use of SDS/PAGE were not used. Instead, Pan et al prepared solutions with zwittergent, which is an ionic solution that prevents irreversible binding of the protein to charged compounds [1] , as well as 3-(N-morpholino)propanesulfonic acid (Mops) and 2-(N-morpholino)ethanesulfonic(Mes) acids were used. Mops and Mes are important because they are good buffering agents that kept solutions to physiological pH. [2] Centrifugation was used to break open the cells, and column filtration using cobalt and cupper beads and imidazole as the elute and phosphate buffer as the wash buffer was used to further purify the prion proteins. Afterwards, SDS-PAGE, immunoblotting, and amino acid analysis (protease degradation) were used to confirm that the proteins were indeed the products. With the purified proteins, FTIR spectroscopy on the amide I' region (1700-1600cm-1) and showed that PrPC had about 42% alpha-helix content and 3% beta-sheet, while PrPSc has 43% beta-sheet and PrP 27-30 has 54% beta-sheet. This was in concordance with the secondary structure predictions using a neural network algorithm, which was done previously. The group then confirmed the high alpha concent f PrPC with circular dichroism studies by recording the ellipticity at 222nm. However, since PrPSc and PrP 27-30 were insoluble, CD was not done. Lastly, EM visualization of the proteins showed that polymers of PrP aggregated into rods while PrPSc and PrPC formed amorphous aggregates. This means the resistant core of the prion protein has the highest amount of LF beta sheet than any of the other two samples studied. From the data described, Pan et al concluded that the formation of PrPSc occurs when the alpha-helices of PrPc convert to beta sheets, and that this conformational change is the main event of the onset of the neurodegenerative diseases. In addition, PrPSc can only affect homologous PrPC proteins, and if the PrPC gene is ablated or mutated enough such that the protein is not made, then the conformational change does not occur. This is important because it negates any possibilities that the PrPSc protein can affect other proteins present in the organism. Furthermore, Pan et al also disproves previous hypothesis that the prion disease can be propagated when prion proteins form amyloids and crystallize. Lastly, Pan et al suggest that somehow the structure and conformation of PrPSc catalyzes its change to an infectious proteins. Also, by identifying the mode of infection of prions, Pan et al distinguish these molecules from other infectious pathogens.


Overall, Pan et al did very meticulous work first on the purification of the proteins in its native form and then on the study of those proteins while maintaining them in its native form. Taking in account of how fragile proteins are, it is very impressive that they are able to take them out of the organism and are able to preserve the proteins. In addition, since the paper did not mention any citations for the exact procedures used to purify the proteins, it is evident that Pan et al did significant work in creating the purification protocol. This purification protocol may also be applied to the purification of other proteins that must be studied in their native forms.

Since the purification procedures described in the paper is a multi-step process, it is imperative that the technique is careful and highly reproducible. One important procedural step Pan et al took was to confirm the presence of the protein in question by SDS-PAGE and the use of anitbodies. This is very important because if at some step in the procedure the proteins are degraded or lost, one will know from performing the checks and can alter procedures that can help to preserve the proteins. Furthermore, the researchers must have had to pay extra attention to make sure that the proteins stay uncontaminated as any unnoticed contaminants may alter the results significantly. For example, the researchers did not know the mechanism of the conformational change. In other words, they did not know whether the change is or can be chemically, thermally, or physically induced. Thus, any chemical contaminant in the protein samples has the potential to alter the conformation of the sample. It is important to note that although it is evident that Pan et al did a lot work to research this topic, they still did not answer many questions about prion proteins, and the discussions in the paper seemed incomplete. For example, they did not discuss the possible modes of how the conformations could have changed-- they simply acknowledged that the change can be chemically induced. However, the change can also by physically or thermally induced. More experiments should have been done to further characterize the nature of the PrPC and the PrPSc and PrP 27-30 core. For example, one possible field of research is to take a handful of the known amino acid residues in the proteins and mutate them either by fluorination or by site-directed mutagenesis. Studying the mutated forms of the proteins and their characteristics can provide insight on what amino acids play a key role in the conformation of the prion proteins. Comparing the conformations of the mutated and native prion proteins may help to unfold the nature of the beta sheet interactions in PrPSc.

Another study can focus on the thermodynamics of protein folding: perhaps Pan et al should also study the energy of formation of the alpha- and beta- rich conformations of the normal versus abnormal versions of the proteins. By doing so, one can gain insight on how spontaneous the conformational change between the isoforms is. Since the spontaneity is based on temperature and concentration, Pan et al can formulate a set of "rules" dictating the maximum concentration of proteins and non-optimal temperature for PrPSc to form insoluble aggregates.

In this lab, Syrian golden hamsters are used to obtain the prion proteins. However, the paper did not discuss the level of homology those prion proteins share with the prion proteins from humans, cows, sheep, and other animals. It seems that the researchers assumed that the hamster prion protein shares enough of a homology to the other prion proteins. This is not always a valid assumption as proteins are usually very specific and the hamster prion proteins may be significantly different that generalizing the same results to the whole family of prion proteins may be wrong. Thus, the researchers should have sequenced the genes of each type of prion protein (i.e. prion protein found in each of the aforementioned organisms) and compared the DNA sequence homology as well as the amino acid sequence homology. This way, any results obtained from studies in the hamsters can be generalized to the other prion proteins, but this generalization must first be weighed by how similar the hamster's prion proteins are to the other types of prion proteins.


Overall, Pan et al did significant work in developing a purification protocol that preserves the purified proteins in its native forms. This is extremely important as most of the protein purification protocols published are written for denaturing conditions. However, although this paper establishes the mode of "infection" of the prion proteins (conformational change between alpha-helix rich to beta-sheet rich protein), it does not even try to explain the mechanism of infection or even the thermodynamic consequences of such a conformational change. In addition, it lacks detail on the structure of the PrPC, PrPSc, and Prp 27-30 proteins in that even though the secondary structure was determined, key amino acid residues and tertiary structures yet remain unknown. In addition, critical assumptions on the homology of the hamster proteins compared to the rest of the prion proteins are made and not proven by either DNA or amino acid sequencing. Thus, this paper lacks necessary experimental data as well as detailed theoretical work, and until further experiments are performed, it is advised that the reader take the paper's claims with skepticism.