How Does Protein Misfolding Occur?

Protein misfolding can occur as a result of a variety of factors. Overproduction of proteins in the cell is a good example. When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein. Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein. This can cause it to misfold into a non-functional state. Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media. Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example, it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.

Why do we care?

Protein misfolding is an important topic in many regards. Many diseases, particularly neurodegenerative disorders such as prion diseases and Alzhemier's disease result from misfolding proteins. The production of recombinant proteins in prokaryotes such as E. Coli can also pose a problem. Non-native proteins are more susceptible to misfolding than native proteins. This can complicate many lab projects such as the design of peptide based drugs. With this in mind, a detection system for protein misfolding could be a very useful tool.

How does our system detect protein misfolding?

Current methods

GFP fusions are a method commonly used to detect protein misfolding. Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase. If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output. If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either, Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system. A more recent system has been the use of a split GFP system. Cabantous et al (2005) describe a system using two fractions of GFP. The smaller part is fused to the target protein. The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest. Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein.

Our System

Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters. By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels. Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell.

Our stress promoters

We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli: Periplasmic Stress Detectors, and one that monitors stress in the cytoplasm of E. Coli: Cytoplasmic Stress Detectors