Extremophile

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== Extraordinary Proteins ==
== Extraordinary Proteins ==
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<StructureSection load='1Y7W' size='350' side='right' caption='Structure of alpha-type carbonic anhydrase (dCAII) (PDB entry [[1y7w]])' scene='JMS/sandbox4/Ca/3'>
 
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== Where there is no man, be a bacteria ==
 
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Where no man or plant could survive, bacteria have been eking out a living, and some even thriving. From the Dead Sea which has 10 times the concentration of salt in salt sea water to the hot springs heated by the molten center of the earth, that pour forth through vents deep under the sea (see this fantastic [http://www.bbc.co.uk/nature/adaptations/Thermophile#p004htvq Thermophile Video] from BBC Wildlife) - in all these hostile environments, life has found footing. To make the question stronger, realize that many things can go wrong, cells could burst or shrivel, DNA can become undone and tattered, protein can unfold into a jumbled mass of amino acids, and membranes made of fat molecules can rip and melt. Environment stress usually achieves all or many of these deadly process to organisms - yet some bacteria survive. To study how the extremophiles (extreme-loving bacteria) survive involves explaining how each of the above processes that should kill the bacteria, in fact do not occur. To understand all of these is a tall order, but to start, in this Proteopedia article, we'll tackle the protein survival under extreme stress problem.
 
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== Extremophiles talk in thermodynamics terms ==
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Life has managed to weather extreme environments - almost every hole we've poked a stick into contains thriving living communities. Proteins are a necessity for living, and therefore tuning protein structures to an extreme environment is of paramount value to an evolving organism. In this article, we present the biophysical modifications present in extreme protein structures.
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All stresses must be interpreted into the language of thermodynamics, since that is the most faithful representation of the problems the environment brings to proteins, and the way the structures have solved this problem, and found a way to maintain their stability.
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The principle equation is:<br />
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== Positively charged myoglobin allows whales to hold their breath during long dives ==
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::<big>∆G = ∆H - T∆S</big>.<br />
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Where ∆G is negative, the movement to products in the reaction is spontaneous. This means, for the case of going from unfolded protein to folded protein as the product, a negative ∆G wold correspond to a stable protein structure. The other three terms: ∆H, T, and ∆S correspond to the change in enthalpy, the temperature (in Kalvins), and the change in entropy. Where the product is more stable than the reactants, ∆H will be negative, and there the produce is more ordered than the reactant, ∆S will be negative. It can be seen from the equation that ∆G becomes more negative for a more negative ∆H or for a less negative ∆S.
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== At the the Weizmann Institute, scientists discover enzymes in the Dead Sea ==
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Elephants can hold their breath for 2 minutes, but whales can hold their breath for 60 minutes - and they do, migrating underwater around the world. To get a clue as to why whales can hold their breath for so long, several researchers gathered tissue samples from hundreds of aquatic and terrestrial mammalian species (mainly from museum collections)<ref name="whaleMyo"> DOI:10.1126/science.1234192</ref>. They measured the concentration of [[myoglobin]], the protein that stores oxygen in muscle tissue, which is used for muscle activity, and also sequenced each specie's myoglobin gene, and used this sequence - as well as the protein's mobility on a native gel (which depends soley on the 3D structure and charge - with myoglobin from different species all having the same overall 3D structure), when possible - to calculate the net charge of each myoglobin protein.
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Our first example comes from the algae Dunaliella salina[[Image:Dunaliella1.jpg|thumb|D. salina]], found growing on the shores of the dea sea by Prof. Volcani, working at the Experimental Research Station (now the Volcani Institute), and then housed on the campus of the Daniel Sieff Institute (now the Weizmann Institute). Appropriately, scientist from the Weizmann Institute, Prof Joel Sussman, Dr. Adi Zamir and collegues, crystallized the <scene name='JMS/sandbox4/Ca/3'>structure of an enzyme</scene> that hangs out of the membrane of D. salina. Now, living on the shore of the Dead Sea presents a special problem for the living. While the dead sea is a challenge with its enormous concentration of salt, living on its shores means one must cope with high salt concentrations, but also - normal salt concentrations. That is, the adaptations to high salt must leave open the possibility of living with normal salt concentrations, too. After comparing the enzyme found on shore dwelling bacteria - appropriately named halo-tolerant (able to withstand high and normal salt concentrations) with enzymes from normal bacteria and purely halophilic ones (some bacteria can even live inside a salt crystal!), they noticed a particular feature of a the protein's structure which was associated with halotolerance. There were <scene name='JMS/sandbox4/Surface/2'>less positively-charged amino acids</scene> - particularly Lysine - on its surface compared to the amount found in the normal enzymes. And unlike the halophilic enzymes, which also had more negatively-charged amino acids, the halotolerant enzyme had a normal number of negatively charged amino acids. Apparently, this intermediate structural property enables the halotolerant enzyme to walk the tightrope, balancing adaptation for high and for medium salt concentrations. Because the thermodynamic threat of salt is not well understood, and nor is the role of increasing the negative surface charge, I label the thermodynamic definition of the structural adaptation ambiguously by not specifying whether it is enthalpic or entropic:<br />
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::<big><scene name='JMS/sandbox4/Ca/3'>∆G</scene> = <scene name='JMS/sandbox4/Surface/2'>∆H-T∆S</scene>.</big><br />
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In the next example, however, I specify the thermodynamic terms which a structural adaptation personifies. For that, we turn to a thermophilic enzyme, also solved by Weizmann Institute lab - Porfessor Yigal Burstein and his team of scientists.
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== Thermophilic enzymes adapt by changing their Enthalpic and Entropic properties ==
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Amazingly, they found that aquatic mammals, across the mammalian phylogeny, independently had acquired the ability to hold their breath, by increasing the concentration of myoglobin, via increasing the net charge of myoglobin. Typically, purified terrestrial mammal's myoglobin has a solubility of 20 mg/g in an aqueous solution at neutral pH ([[http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/m0630pis.pdf Sigma Aldrich]]) which turns out to be the maximum level of myoglobin found in most terrestrial mammal's tissue. But whales and other aquatic mammals far exceed this solubility limit, e.g., whales have 70 mg/g. The way that they overcome the solubility constraint may be traced back to a modest increase in the net charge of myoglobin - from around +2 in terrestrial animals to around +4 in aquatic animals.
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Some bacteria and even animals can survive great temperatures. Studying <scene name='JMS/sandbox5/Tbadh/1'>a thermophilic enzyme</scene>, Pof Burstein noticed two special features that appear to explain this enzymes ability to maintain its structure in over 83℃! For comparison, you could fry an egg at 65℃, which mean all the protein in an egg denature at significantly less than 83℃. To demonstrate the special structural properties of the thermophilic enzyme underlies its thermophilic prowess, Prof Burstein selectively altered normal enzymes to have the two structural features, and indeed found that the normal enzymes had become thermophilic. The two properties relate to ∆H and to ∆S. Firstly, he found the thermophilic enzyme had a unique <scene name='JMS/sandbox5/Ion_network/4'>four amino acid binding-network</scene> that encompassed two monomers of the tetrameric enzyme, repeating between each monomer and its two partner monomers. This network apparently makes the oligomer more stable, or ∆H more negative. Secondly, the thermophilic enzyme was <scene name='JMS/sandbox5/Proline/2'>enriched for proline</scene>. Because proline's residue (side chain) has no, or minimal, possible degree of freedom, therefore proline's side chains, unlike other amino acids - is not restricted by folding. Therefore, ∆S is less negative. These two structural properties are labelled by the corresponding term in the thermodynamics equation:<br />
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::<big><scene name='JMS/sandbox5/Tbadh/1'>∆G</scene> = <scene name='JMS/sandbox5/Proline/2'>∆H</scene> - T<scene name='JMS/sandbox5/Ion_network/4'>∆S</scene>.</big><br />
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However, a 3-fold increase in concentration of myoglobin ought to result in a similar fold increase in max time of breath holding, and the researchers show that body mass also makes a critical contribution to an animal's ability to hold its breath, with the overall equation for the contribution of body mass and myoglobin net charge as follows:
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log (maximum time underwater) = 0.223*log(body mass) + 0.972*log(myoglobin net charge) + 0.891
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As Asian elephant's weight is ~3K Kg, and a sperm whale's weight is ~50K Kg, it is clear that the modest increase in net charge contributes about the same as the enormous difference in body mass to the maximum time underwater.
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<StructureSection load='1mbn' size='350' side='right' caption='myoglobin (PDB entry [[1mbn]])' scene='55/557585/Align_test/5'>
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==Molecular Tour==
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The ability of increasing net charge to enable higher solubility is a known phenomenon<ref>doi: 10.1073/pnas.0402797101</ref>, and this study is consistent with previous reports<ref>PMID: 14741208 </ref>. The aquatic animals have increased their net charge in a variety of ways - different combinations of amino acids switches. We present one such manifestation of this overall trend, by comparing the elephant and whale myoglobin structures.
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It comes down to <scene name='55/557585/Align_test/18'>eight divergent amino acids (elephant's amino acids in yellow halos, and whale's amino acids without yellow halos, next to each other)</scene> that affect that charge - out of a total of 27 divergent amino acids. Without these eight differently charged amino acids, myoglobin in both whale and elephants has a charge of ''+1''. With them, whale myoglobin has a net charge of ''+4'' and elephants of ''+2''. Importantly, all eight of these divergent amino acids are <scene name='52/523344/Elephantwhale/34'>surface residues</scene>. It is interesting to note that in this comparison (between whale and elephant) that there is no difference in net charge in the region in the vicinity of the heme group. This may reflect the key role of the heme group and residues near it, which can not be easily changed without a drastic affect on function.
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Calculate along the chain, how just several amino acid switches bring the positive net charge of whale myoglobin up to ''+4'' and elephants to ''+2'': (summing up the total charge of the protein) <scene name='52/523344/Elephantwhale/19'>residue position 8</scene> (<span style="color:red">'''glu'''</span> in elephents versus gln in whales), <scene name='52/523344/Elephantwhale/21'>12</scene> (<span style="color:blue">'''lys'''</span> vs. <span style="color:lightblue">'''his'''</span>), <scene name='52/523344/Elephantwhale/22'>27</scene> (thr vs. <span style="color:red">'''asp'''</span>), <scene name='52/523344/Elephantwhale/23'>34</scene> (thr vs. <span style="color:blue">'''lys'''</span> ), <scene name='52/523344/Elephantwhale/24'>87</scene> (gln vs. <span style="color:blue">'''lys'''</span> ), <scene name='52/523344/Elephantwhale/26'>116</scene> (gln vs. <span style="color:lightblue">'''his'''</span>), <scene name='52/523344/Elephantwhale/27'>132</scene> (<span style="color:blue">'''lys'''</span> vs. asn), <scene name='52/523344/Elephantwhale/28'>140</scene> (asn vs. <span style="color:blue">'''lys'''</span> ).
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'''note about this scene''': <span style="color:red">'''Asp and Glu'''</span> have a charge of ''-1'', <span style="color:blue">'''Arg and Lys'''</span> have a charge of ''+1'', <span style="color:lightblue">'''His'''</span> in the positions shown here - ''12'' and ''116'' (Table S2<ref name="whaleMyo" />) - have a charge of about ''+0.5''.
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</StructureSection>
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{{Reflist}}

Current revision

Extraordinary Proteins

Life has managed to weather extreme environments - almost every hole we've poked a stick into contains thriving living communities. Proteins are a necessity for living, and therefore tuning protein structures to an extreme environment is of paramount value to an evolving organism. In this article, we present the biophysical modifications present in extreme protein structures.

Positively charged myoglobin allows whales to hold their breath during long dives

Elephants can hold their breath for 2 minutes, but whales can hold their breath for 60 minutes - and they do, migrating underwater around the world. To get a clue as to why whales can hold their breath for so long, several researchers gathered tissue samples from hundreds of aquatic and terrestrial mammalian species (mainly from museum collections)[1]. They measured the concentration of myoglobin, the protein that stores oxygen in muscle tissue, which is used for muscle activity, and also sequenced each specie's myoglobin gene, and used this sequence - as well as the protein's mobility on a native gel (which depends soley on the 3D structure and charge - with myoglobin from different species all having the same overall 3D structure), when possible - to calculate the net charge of each myoglobin protein.

Amazingly, they found that aquatic mammals, across the mammalian phylogeny, independently had acquired the ability to hold their breath, by increasing the concentration of myoglobin, via increasing the net charge of myoglobin. Typically, purified terrestrial mammal's myoglobin has a solubility of 20 mg/g in an aqueous solution at neutral pH ([Sigma Aldrich]) which turns out to be the maximum level of myoglobin found in most terrestrial mammal's tissue. But whales and other aquatic mammals far exceed this solubility limit, e.g., whales have 70 mg/g. The way that they overcome the solubility constraint may be traced back to a modest increase in the net charge of myoglobin - from around +2 in terrestrial animals to around +4 in aquatic animals.

However, a 3-fold increase in concentration of myoglobin ought to result in a similar fold increase in max time of breath holding, and the researchers show that body mass also makes a critical contribution to an animal's ability to hold its breath, with the overall equation for the contribution of body mass and myoglobin net charge as follows:

log (maximum time underwater) = 0.223*log(body mass) + 0.972*log(myoglobin net charge) + 0.891

As Asian elephant's weight is ~3K Kg, and a sperm whale's weight is ~50K Kg, it is clear that the modest increase in net charge contributes about the same as the enormous difference in body mass to the maximum time underwater.

myoglobin (PDB entry 1mbn)

Drag the structure with the mouse to rotate
  1. ↑ 1.0 1.1 Mirceta S, Signore AV, Burns JM, Cossins AR, Campbell KL, Berenbrink M. Evolution of mammalian diving capacity traced by myoglobin net surface charge. Science. 2013 Jun 14;340(6138):1234192. doi: 10.1126/science.1234192. PMID:23766330 doi:http://dx.doi.org/10.1126/science.1234192
  2. ↑ Brocchieri L. Environmental signatures in proteome properties. Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8257-8. Epub 2004 May 24. PMID:15159533 doi:http://dx.doi.org/10.1073/pnas.0402797101
  3. ↑ Goh CS, Lan N, Douglas SM, Wu B, Echols N, Smith A, Milburn D, Montelione GT, Zhao H, Gerstein M. Mining the structural genomics pipeline: identification of protein properties that affect high-throughput experimental analysis. J Mol Biol. 2004 Feb 6;336(1):115-30. PMID:14741208 doi:http://dx.doi.org/10.1016/S0022283603014748
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