Abstract

Methane clathrates along continental margins and in permafrost store thousands of gigatons of carbon in sediments and serve as a habitat for a unique deep subsurface biosphere. We hypothesized that clathrate-dwelling microbes utilize clathrate-binding proteins (CBPs) that enable them to inhabit the extreme environment of methane clathrates in a manner analogous to antifreeze proteins (AFPs). We identified a number of candidate CBPs, or CbpAs, in metagenomes from gas clathrate-bearing sediment that contain the TxxxAxxxAxx motif found in canonical Type I AFPs from cold water fish; this motif enables Type I AFPs to inhibit gas clathrate growth. Recombinantly expressed CbpAs were shown to bind to and alter the morphology of tetrahydrofuran (THF) clathrate, a structure II clathrate that is stable at atmospheric pressures and 4˚C. Importantly, CbpAs inhibit the growth of biologically relevant methane clathrate, and are as effective at inhibition as commercial clathrate inhibitors. Structural characterization of CbpA₃ and molecular dynamics simulations revealed an unprecedented protein fold and clathrate-binding mechanism different from Type I AFPs. The discovery of clathrate-binding proteins in gas clathrate-bearing sediments has many implications for climate change, natural gas pipeline flow assurance and transport, microbial survival strategies, and the search for life on other planetary bodies.