In our Protein Journal Club this semester, we are studying proteins from extremophiles. As their name suggests, extremophiles are organisms that can survive under extreme conditions. These extreme conditions include acidic or basic environments, severe hot or cold environments, lack of oxygen, high salt concentrations, high sugar concentrations, high hydrostatic pressure, high levels of ionizing radiation, high concentrations of heavy metals, and extremely dry conditions such as those found in deserts.
I’m a big fan of sci-fi shows because they ask the questions “What if there were life on another planet?” or “What if we were to colonize another planet?” But these questions don’t just have to be answered in the form of fiction. We have extreme conditions on Earth which simulate those found on other planets such as Mars. If life exists on Mars, it is likely to be able to survive extremely cold temperatures, for example. Also, if we want to eventually establish a colony on Mars, we have to be able to adapt plants to grow in extreme cold conditions. These are a few of the reasons I am interested in psychrophiles, organisms able to survive and grow in temperatures as low as -15 °C.
The protein I am focusing on is α-amylase from the organism Alteromonas haloplanctis, found in Antarctic seawater, which maintains a constant temperature between -2 and +2 °C. It is the first psychrophilic protein to be crystallized1 and therefore has been extensively studied and compared to similar proteins from mesophiles (organisms which grow in moderate temperature) and thermophiles (organisms which grow in extreme heat).
Proteins from psychrophiles have lower melting temperatures (temperatures at which half the protein is unfolded) than those from mesophiles and thermophiles.3 This means they are less stable. However, at the low temperatures at which these organisms grow, their proteins are stable enough to carry out their function. This sacrifice of stability is not without benefit: most proteins are inactive at low temperatures, but these psychrophilic enzymes retain their activity in the cold.
What changes in the structure of psychrophilic proteins cause them to lose stability but gain activity? There are many ways this can occur, but most involve increasing the flexibility of the protein. For A. haloplanctis α-amylase, there are fewer charged amino acids overall and especially at the surface of the protein.2 Additionally, there are fewer proline residues (this amino acid adds rigidity to proteins because its side chain has a cyclic structure connected to the backbone in two places rather than one). There are also fewer inter-domain interactions. A structurally important calcium ion and a catalytically important chloride ion are both held less tightly, and fewer helices are stabilized at their N- and C- terminal ends. All of these things add up to mean that A. haloplanctis α-amylase is more flexible, which raises the energy of the initial enzyme-substrate complex and allows it to have a higher reaction rate by lowering its overall activation energy (Figure 2).
Researchers have used a computer program called PoPMuSiC to calculate changes in stability for each amino acid residue if it were to be replaced with all other amino acids.4 For example, a serine to alanine mutation at a particular residue could result in a change in energy of -0.5 kcal/mol. They determined that overall, most mutations in α-amylase are destabilizing. Interestingly, the catalytic domain A of the psychrophilic protein was determined to be slightly more stable than that of its mesophilic counterparts, whereas domains B and C were less stable in the psychrophilic protein. Using the calculated changes in stability for each mutation, they proposed several mutations that should stabilize the psychrophilic protein. Such mutations in psychrophilic α-amylase have been shown to raise the melting temperature and confer similar activity to that of mesophiles.3
1. Aghajari N., Haser R., Feller G. & Gerday C. (1998). Crystal structures of the psychrophilic α-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor, Protein Science, 7 (6) 1481-1481. DOI: 10.1002/pro.5560070626
2. Aghajari N., Feller G., Gerday C. & Haser R. (1998). Structures of the psychrophilic Alteromonas haloplanctis α-amylase give insights into cold adaptation at a molecular level, Structure, 6 (12) 1503-1516. DOI: 10.1016/S0969-2126(98)00149-X
3. Feller G. (2010). Protein stability and enzyme activity at extreme biological temperatures, Journal of Physics: Condensed Matter, 22 (32) 323101. DOI: 10.1088/0953-8984/22/32/323101
4. Gilis D. (2006). In Silico Analysis of the Thermodynamic Stability Changes of Psychrophilic and Mesophilic α-Amylases upon Exhaustive Single-Site Mutations., ChemInform, 37 (31) DOI: 10.1002/chin.200631215