Structural and Functional Analysis of Ice – Nucleation Proteins (INP) from the strain Pseudomonas syringae R10.79

Abstract

Ice Nucleation Proteins (INP) are integral membrane proteins that aid in forming ice crystals. As the name suggests INP help form ice crystals by forming the nucleus around which water molecules align themselves. Under favourable conditions, these water molecules freeze when associated with the protein. INP are present in numerous organisms, the focus of this project, however, was INP from the bacteria – Pseudomonas syringae R10.79. Evidence from a previous study suggested that the number of repeats present in the Central Repeat Domain (CRD) directly affects the Ice Nucleation (IN) activity and the IN temperature. Truncated versions of the INP was compared for their IN activity and IN temperature. The repeats in the CRD were distinct from one another in some specific amino acid residues. In this project we designed INP with 9 and 16 repeats (R), identical in their amino acid composition. We wanted to analyse if a more uniform and identical CRD increases the ice nucleation activity. Vectors containing the gene of interest were transformed into different E. coli protein expression strains. The proteins contained a poly-His tag attached to the N-terminal of the INP. After successive troubleshooting, a Western blot analysis of the protein product obtained showed the absence of a His-tagged protein. Sequence analysis of the vector constructs revealed a very high number of the rare codons for Arg, which could be a reason behind problems in production of the protein in E. coli due to codon bias. Another set of INP with 9 repeats and 16 repeats with distinct repeats, that showed effective IN activity was also worked on. With these repeats the goal was to achieve a protein of high purity. This would enable the structural characterisation of the proteins by X-ray crystallography. Three different rounds of protein purification (affinity, ion-exchange and size-exclusion chromatography) resulted in a protein with a very high purity.

Popular Abstract

Cloudy with a chance of Bacteria

When we look up into the sky we see shapes of white, floating across as clouds. We know clouds are the source of rain, snow and hail. But, did you ever imagine that bacteria might be one of the contributors to precipitation? One such bacteria is, Pseudomonas syringae. It travels from plant surfaces up into the atmosphere, and into clouds. These bacteria are known to help in the special arrangement of water molecules that helps creating ice crystals. These crystals form around a special protein present on the surface of the bacterial cell – the Ice-Nucleation Active Protein or INP. The basis of this study was the analysis of the structure and function of the INP.

The structure of INPs contains 3 main parts:
(i) a water repelling part (N-terminal), (ii) a water loving part (C-terminal), and (iii) a highly conserved central part (Conserved Central Region - CCR). CCR is important in binding water molecules and is known to contain 60-80 amino acid repeating units that are, slightly, distinctive from one another. It has the ability to freeze water molecules at temperatures close to 0°C, much higher than the usual freezing temperature of pure water that is - 36°C. Other substances that are capable of promoting ice crystal formation are: dust, soot, etc. But they do so only at much lower temperatures -20°C or lower. To check the significance of the size of the CCR for the ice-nucleation activity, the CCR was reduced by eliminating some repeating units. It was found that shortening the CCR affected the temperature at which water molecules would freeze. A shorter CCR caused freezing at much colder temperature than the longer CCR.

To analyse the role that the exact sequence of an amino acid repeat plays for the activity of INP, we produced an INP that contained only repeats with perfect sequence. This is the sequence that we think is responsible for ice formation. There were 2 versions of the INP containing 9 repeats (INP_9R) and 16 repeats (INP_16R). The repeats were identical in their composition of amino acid residues. Such an INP hadn’t been expressed before. The protein was tried to be produced in a bacterium that is specialized in large-scale protein production. Protein production was unsuccessful and the reasons for this could be the repetitive nature of the CRD in the protein. Further another shortened version of the INP was modelled, that also contained 9 repeats and 16 repeats, however, in this case the repeats were not identical. These proteins were successfully produced in the bacterial expression system, and a highly purified protein product was obtained.In the future, we would like to look into the physical structure of the proteins. We would also like to see how the structure of the INP is related to its sequence and affects freezing. We will do this by visualising the protein molecules using electron microscopy and also determine the structuralfeatures by X-ray crystallography. Characterising the integral parts of the protein and the role they play in freezing of water will help interpret their impact on the water cycle, and other effect it has on the ecosystem.

Master’s Degree Project in Molecular Biology, specialisation in Microbiology, 45 credits, 2019,
Department of Biology, Lund University
Advisor: Kai Finster, Tina-Santl Temkiv, Thomas Boesen
Department of Biosciences, Aarhus University, Denmark

• author: Chandra, Urvshi Kaveri
• supervisor: Kai Finster
• organization: Degree Projects in Molecular Biology
• course: MOBN02 20181
• year: 2019
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8995239
• date added to LUP: 2019-09-17 11:56:05
• date last changed: 2019-09-17 11:56:05