Tag Archives: antifreeze proteins

The humble snow flea teaches us some protein chemistry

Who would have thought that the humble snow flea (that we used to cross country ski over in Montana) would teach us a great deal about protein chemistry turning over some beloved shibboleths in the process.

The flea contains an antifreeze protein, which stops ice crystals from forming inside the cells of the flea in the cold environment in which it lives. The protein contains 81 amino acids, is 45% glycine and contains six  type II polyProline helices each 8 amino acids long (https://en.wikipedia.org/wiki/Polyproline_helix). None of the 6 polyProline helices contain proline despite the name, but all contain from 2 to 6 glycines. Also to be noted is (1) the absence of a hydrophobic core (2) the absence of alpha helices (3) the absence of beta turns (4) the protein has low sequence complexity.

Nonethless it quickly folds into a stable structure — meaning that (1), (2), and (3) are not necessary for a stable protein structure. (4) means that low sequence complexity in a protein sequence does not invariably imply an intrinsically disordered protein.

You can read all about it in Proc. Natl. Acad. Sci. vol. 114 pp. 2241 – 2446 ’17.

Time for some humility in what we thought we knew about proteins, protein folding, protein structural stability.

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Curioser and curioser

Curious Wavefunction alluded to the first example of a protein which stands everything we thought we knew about them on its head. At the end of this post you’ll find another equally counterintuitive example.

We all know that proteins fold into a relatively dry core where hydrocarbon side chains and other hydrophobic elements hide out. This was one of Walter Kauzmann’s many contributions to chemistry and biology. He also wrote one of the first books on quantum chemistry, as did his PhD advisor Henry Eyring at Princeton (I was lucky enough to take PChem from him). The driving force for the formation of globular proteins according to him, was pretty much entropic, with hydrocarbon side chains solvating each other so water wouldn’t have to form an elaborate (hence structured) cage to do so.

Which brings us to the wonderfully named fish Pseudopleuronectes Americanus which lives in frigid polar waters. To keep ice crystals from forming in their cells, arctic fish have evolved proteins to prevent it. It is a fascinating example of evolution solving a problem different ways, because by 1996 at least 4 different types of antifreeze proteins were known [ PNAS vol. 93 pp. 6835 – 6840 ’96 ].

The new protein is a 3 kiloDalton alanine rich helix bundle 145 Angstroms long.
Amazingly the helices surround a core of 400 water molecules (surround as in the water is on the inside of the protein, not the outside). The water molecules inside the protein are arranged as pentagons (not hexagons as they would be in ice) — so they form a clathrate. The pentagonal arrangement of water was predicted on theoretical grounds 50 years ago by Scheraga ( J. Biol. Chem. vol. ?? pp. 2506 – 2508 1962 ).

The protein has an amino acid periodicity of 11 amino acids, which nicely comes out to 3 turns of the alpha helix. There is a threonine at position i, alanine at position i + 4 and alanine a position i + 8. All of these bind water — not surprising for threonine, but alanine is a hydrocarbon. The evolving fish clearly didn’t listen to protein chemists. However, most of carbonyl groups of the protein backbone are involved in hydrogen bonding to water.

Not to be outdone, a freeze tolerant beetle (Upis cermaboides — don’t you love these names) has an antifreeze molecule made mostly of sugar and lipid.

Well even if we don’t know what we thought we knew about proteins, at least we understand biologic membranes and the proteins that go through them. Don’t we?

Apparently not. [ Proc. Natl. Acad. Sci. vol. 111 pp. 2425 – 2430 ’14 ] studied the alpha-hemolysin of staphylococci. We know that the membrane of our cells is made of a double layer of molecules which a charged head which binds water and a long (16 + carbons) hydrocarbon tail. So the hydrocarbon core is 30 Angstroms across, and the lipid head groups are about 40 Angstroms away from each other on either side of the membrane.

We also know how proteins fit into the membrane — one model is the G Protein Coupled Receptor (GPCR) for which we have at least 800 human genes, and which is the target for 30% of all drugs approved by the FDA [ Science vol. 335 pp. 1106 – 1110 ’12 ]. These all have 7 alpha helices arranged like a stack of logs extending across the membrane. The amino acids here are usually hydrophobic. Another model is the beta barrel — used mostly by bacteria — these have beta strands arranged across the membrane (like the staves of a barrel — get it). I’m not sure what the record is for the number of strands, but one from the gonococcus has 16 of them. They surround a large pore.

Back to the alpha hemolysin of staphylococci It’s designed to kill its target by forming a hole in the membrane. And so 7 of them get together to do so. However, instead of the running back and forth across the 30 Angstroms of the anhydrous part of the membrane, the heptamers put their heads together forming the hole (like skydivers holding hands), with their hydrocarbon like parts sticking out into the membrane and the water filled hole in the center. How do they know? They studied truncated mutants of the hemolysin, which weren’t long enough to span the 30 Angstroms across the membrane, and they still formed holes. An entirely new (to me) protein arrangement.