From serendipity to precision: investigating pH-induced oligomerization switch for protein control

Abstract

Understanding the pH sensitivity of proteins and enzymes across intracellular compartments is crucial for fundamental research. Lizatović et al. [1] aimed to design a peptide incorporating a specific amino acid to form a hexamer structure and lacking it to form a pentamer structure. Unexpectedly, two distinct structures emerged based on pH. At pH 6, a hexamer structure formed, albeit with a surprising arrangement, while at pH 8, a pentamer structure appeared despite the presence of the specific amino acid. This peptide came to be abbreviated as pHios (pH-induced oligomerization switch).

The pH-induced oligomerization switch prompts several questions: is it feasible to use the designed peptide to regulate the structure and function of other proteins within a tightly controlled pH range? This would involve fusing pHios with the desired protein to exploit its pH-induced oligomerization capability to modulate the structure and activity of the fused protein. Can new sequences with similar properties be rationally designed? Will slight alterations to the designed sequence still maintain functionality?

Experimental methods, including size exclusion chromatography (SEC) to assess protein size changes, and circular dichroism (CD) to study secondary structure alterations, were employed on redesigned sequences of the wild-type (WT) peptide at varying pH levels. Results indicate limited tolerance to sequence modifications, although cautious interpretation is warranted due to suboptimal fitting and low concentrations. Certain sequences exhibit differing stability at pH 6 or pH 8. The fusion of pHios to the target protein faced expression challenges, highlighting the need for optimized protein expression protocols.

Popular Abstract

pH power: exploring the impact of protein shape and activity

A cell is the smallest living unit in all organisms. It works like a tiny factory, producing energy, storing information, and performing essential functions to sustain life. Cells consist of different compartments with varying pH levels. Proteins, essential molecules inside these compartments, perform numerous vital functions and are composed of chains of building blocks (amino acids), similar to beads on a necklace.

Researchers have previously worked on designing a small protein intended to have a specific shape, where long helical threads coil around each other. In nature, such structures typically have 3, 4, or 5 threads, but the researchers attempted to create one with 6. The design intended to result in 6 threads with a specific amino acid present, and 5 threads without it. However, the outcome was different from expectations. Two distinct structures were observed depending on pH. At pH 6, a structure with 6 threads formed, but their arrangement was unexpected. At pH 8, a structure with 5 threads appeared, despite the specific amino acid being present.

The discovery led to new questions: can the designed protein and its pH-dependent structural changes be used to modify the shape and activity of another protein? By attaching the designed protein to the target, could we control its activity by forcing it into the desired structure at a specific pH? Another question is how “perfect” this designed system is. Can small modifications to its building blocks still maintain its functionality? To investigate this, two experimental methods are used. One method aims to determine how the size of the protein’s changes. The other method studies how their shapes change.

If the designed protein can alter other protein’s structure and function via pH, it could open up many possibilities. For example, genes in our bodies serve as blueprints for how the body should be built and function, and faulty genes can lead to illness. By leveraging the designed protein and the variation in pH across different cell compartments to control the opening of specialized containers made up of protein molecules, we can use them as vehicles to deliver the components needed to repair the faulty genes.

Our study found that the system doesn’t adapt well to small changes in its building blocks. Some redesigned proteins were more stable at pH 6, while others preferred pH 8. When we tried to combine the designed protein with a target protein, we had trouble producing it, highlighting the need for better protein production methods.

• author: Bäckström, Isabelle
• supervisor: Ingemar André
• organization: Department of Chemistry
• course: KEMR10 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Biochemistry, Oligomerization, pH
• language: English
• id: 9164989
• date added to LUP: 2024-06-20 14:33:09
• date last changed: 2024-06-20 14:33:09