Sticking to the story at the molecular level

A deeper understanding of protein adhesion to solid surfaces may shed new light on biological phenomena such as marine biofouling. In their quest for non-toxic, microorganism-repelling surfaces, Singapore's Agency for Science, Technology and Research (A*STAR) researchers evaluated the relationship between charge and pH for an adhesive protein that exists in minute quantities in the footprint of barnacle larvae and showed it influences their ability to attach to surfaces.

The measurements led to a specific pH value known as isoelectric point (pI), at which the protein net charge equals zero. The pI provides pH ranges for protein solubility, but also gives valuable information on a protein's affinity to charged surfaces, which is essential for protein separation, sensing, and nonspecific adsorption. Proteins lose or gain protons depending on the acidity of their surroundings, which alters their net charge. They typically present a positive net charge under highly acidic conditions and a negative charge in highly basic environments. This enables attractive interactions with oppositely-charged substances. At pI, the zero net charge promotes protein aggregation.

Several approaches to determine pI values already exist but tend to be time-consuming and require high water solubility and concentration. Under the Innovative Marine Antifouling Solutions program, a team led by Julius Vancso, from the A*STAR Institute of Chemical Engineering Sciences, and Dominik Jańczewski, from the A*STAR Institute of Materials Research and Engineering, has developed a strategy that delivers protein pI values using atomic force microscopy (AFM).

"AFM has become an enabling platform to visualize as well as manipulate and study matter at a molecular scale," explains Vancso, noting that his team has used AFM to investigate macromolecular behavior for about 25 years. The researchers anchored a few protein molecules to AFM probes approximating one micrometer across and assessed their adhesion force to charged surfaces at well-defined pH values while pulling the probes off these surfaces.

After validating this approach for various well-known proteins, the team tackled the pI value of the footprint protein. According to Vancso, this protein stimulates the attachment of a larva, which triggers colonization and further build-up by other larvae. The protein exhibited a pI value of 9.6 to 9.7, consistent with its positive charge in seawater and its adhesiveness to the negatively charged immersed surfaces.

This proof-of-concept experiment minimized protein amount requirements. "We hope that we contributed to the solving of this notoriously difficult and very essential issue," says Vancso. They expect that protein scientists will adopt their technique.

The A*STAR-affiliated researchers contributing to this research are from the Institute of Chemical Engineering Sciences and Institute of Materials Research and Engineering.