Mainly on the Plane: Deep Subsurface Bacterial Proteins Bind and Alter Clathrate Structure

Abstract

Gas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a low-pressure analogue for structure II gas clathrate. When grown in the presence of CbpAs, the THF clathrate was polycrystalline and platelike instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a platelike structure during gas clathrate transportation.

Molecular basis for inhibition of methane clathrate growth by a deep subsurface bacterial protein

“…In this study, we report bacterial clathrate-binding proteins (CbpAs) that potently inhibit methane clathrate growth. These bacterial CbpAs were initially discovered in metagenomes from deep subsurface methane clathrate-bearing sediments based on their threonine-alanine (TxxxAxxxAxx) motifs common to AFPs ( 16 ). Recombinantly expressed and purified CbpAs bind to and alter the morphology of structure II tetrahydrofuran clathrate, a low-pressure analog for gas clathrate ( 16 ).…”

“…These bacterial CbpAs were initially discovered in metagenomes from deep subsurface methane clathrate-bearing sediments based on their threonine-alanine (TxxxAxxxAxx) motifs common to AFPs ( 16 ). Recombinantly expressed and purified CbpAs bind to and alter the morphology of structure II tetrahydrofuran clathrate, a low-pressure analog for gas clathrate ( 16 ). Here, we show that these proteins suppress methane clathrate growth and we then provide a molecular basis for this activity through structural and biochemical studies combined with molecular dynamic (MD) simulations.…”

Adsorption‐Inhibition of Clathrate Hydrates by Self‐Assembled Nanostructures

“…[23,46] Recently, the Glass group identified a new class of proteins that bind to the surface of THF hydrates. [47] These clathrate-binding proteins (CBDs) were identified in DNA sequences of bacteria from hydrate-rich areas and possess high sequence homology to the alanine-rich AFPI. [47] A small dye molecule, safranine O (SFO), was found to inhibit the growth of ice crystals at millimolar concentrations.…”

“…[47] These clathrate-binding proteins (CBDs) were identified in DNA sequences of bacteria from hydrate-rich areas and possess high sequence homology to the alanine-rich AFPI. [47] A small dye molecule, safranine O (SFO), was found to inhibit the growth of ice crystals at millimolar concentrations. [48] The presumed mechanism of inhibition, based on metadynamics simulations and SFO crystal structure, is the formation of a supramolecular assembly that resembles the structure of AFPs.…”

What is the role of microbes in gas hydrate formation and stability?

“…Some amino acids function as hydrate promoters, while others are inhibitors (Bavoh et al, 2019; Bhattacharjee & Linga, 2021). Recombinantly expressed clathrate‐binding proteins (CbpAs) encoded in genomes of hydrate bacteria bind to and change the morphology of tetrahydrofuran hydrate (Johnson et al, 2020) and methane hydrate (Huard et al, in review). The activity of proteins as hydrate inhibitors has also been demonstrated in natural samples from a hydrate deposit in the South China Sea (Liu et al, 2021).…”