Difference between revisions of "Team:CLSB-UK/Project/CmpA"

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<h2> CmpA Gene </h2>
 
<h2> CmpA Gene </h2>
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<h4> Overview </h4>
 
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CmpA encodes the substrate-binding protein of the HCO<sub>3</sub><sup>-</sup> transporter and plays a crucial role in the carbon-concentrating machinery of synechocystis, necessary for efficient energy production via photosynthesis. CmpA has never been made into a Biobrick, and the CLSB UK team synthesized and then characterized this part in our model organism. Despite it having the highest affinity for Bicarbonate and Carbonic acid in the Synechocystis genome it is the rate limiting step of CO2 absorption in the CmpAD operon, so in theory to overexpress it would increase the rate of growth of Synechocystis, especially important as the current slow rate of growth is one of the largest issues with using Synechocystis as a model organism.
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The CmpA gene forms the first part of the CmpABCD operon, which codes for a bicarbonate transporter. It encodes the substrate-binding protein of the transporter and plays a crucial role in the carbon-concentrating machinery of synechocystis. 
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<h4> The Operon: </h4>
  
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<p>
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CmpA forms the first part of the CmpABCD operon, coding for proteins that allow dissolved Carbon Dioxide to be transported into cyanobacteria for use in photosynthesis. CmpA is the substrate binding membrane protein, CmpB is an integral channel protein, and CmpC and D are ATPases to drive the active transport, as well as CmpC controlling the transport rate.
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The operon is part of the CO<sub>2</sub> concentrating mechanism (CCM) that allows CO<sub>2</sub> to be concentrated up to 1000 times around Rubisco, an essential enzyme which catalyses a reaction with CO<sub>2</sub> in photosynthesis and is inhibited by oxygen. Since the Great Oxidation Event, the inhibition of Rubisco by Oxygen caused Rubisco to be very inefficient, and the concentration of CO<sub>2</sub> in the Carboxysome results in a significant increase in photosynthetic efficiency. As cyanobacteria are water dwelling, CO<sub>2</sub> is transported in its dissolved form – H<sub>2</sub>CO<sub>3</sub> or HCO<sub>3</sub><sup>-</sup> depending on the pH. This is converted to CO<sub>2</sub> in the carboxysome.
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CmpA has the highest affinity to both the bicarbonate ion and carbonic acid of any protein in the cyanobacteria genome. It is used to transport bicarbonate ions (HCO<sub>3</sub><sup>-</sup> ) into the CmpB channel.
 
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<span class="label"><b>Figure 1.</b> Structure of CmpA-D in the membrane</span>
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<b>Function</b>
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<h4>Structure</h4>
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CmpA is an α-ß protein similar in structure to the nitrate transporter NrtA. It consists of two domains arranged in a C-clamp shape around the ligand-binding site. Both of these domains contain a central complex of five-stranded ß-pleated sheets surrounded by α-helices. They are joined to each other by a sheaf of irregularly coiled elements. The entire protein is anchored to the plasma membrane by a lipid; CmpA is a member of the periplasmic-binding protein superfamily.  
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The CmpA protein is used to transport bicarbonate ions (HCO3-) into the carboxysome, an organelle that contains Rubisco. The HCO3- is then converted into CO2. This mechanism allows synechocystis to concentrate CO2 in the carboxysome around Rubisco and exclude O2, for which Rubisco also has a high affinity. The concentration of CO2 inside the carboxysome can be up to 1000 times higher than in the cytoplasm, resulting in a significant increase in photosynthetic efficiency.  
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<h4>Mechanism</h4>
  
 
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Bicarbonate ions bind to the ligand-binding site contained within the cleft formed by the two α-ß domains. These two domains then wrap around the ligand. This involves only minor conformational changes; the ligand-binding site is fairly inflexible. Above is a ribbon diagram showing CmpA at pH 5.0 on the left and 8.0 on the right. The bicarbonate ion is shown on the left in the entrance to the cleft as a sphere. The carbonic acid is shown on the right deep inside the cleft as a sphere. Carbonic acid can only bind in the presence of Ca<sup>2+</sup> ion, which may act as a cofactor and is also shown as a sphere on the right.
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<b>Structure</b>
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<h4>Genetic Approach</h4>
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Given the <a href="http://genome.microbedb.jp/cyanobase/Synechocystis/genes/slr0040"> availability of this gene's sequence</a>, the most sensible approach was to design the primers for amplifying this specific DNA directly out of the Synechocystis PCC sp. 6803 genome. So, initially, we set about designing the pair of primers that would allow us to amplify this specific gene. Unfortunately, having looked at different options for this, we encountered a significant problem. The primers that would have given us the most useful stretch of DNA would have, most likely, formed primer dimers.
  
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CmpA is an α-ß protein similar in structure to the nitrate transporter NrtA. It consists of two domains arranged in a C-clamp shape around the ligand-binding site. Both of these domains contain a central complex of five-stranded ß-pleated sheets surrounded by α-helices. They are joined to each other by a sheaf of irregularly coiled elements. The entire protein is anchored to the plasma membrane by a lipid; CmpA is a member of the periplasmic-binding protein superfamily.  
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As a result, we have opted for having the gene synthesized as a gBlock by IDT. This approach turned out to be much better as we could synthesise the gBlock that contained the RBS and BioBrick prefix and suffix, simplifying our cloning strategy. We chose to use the RBS that was described in Wang et al. 2012. We then amplified this construct using the oligo primers that we also had synthesised by IDT.
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<img src="https://static.igem.org/mediawiki/2016/5/59/T-CLSB-UK--cmpA.png">
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<span class="label"><b>Figure 1.</b>Structure of the CmpA protein.</span>
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<b>Mechanism</b>
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Bicarbonate ions bind to the ligand-binding site contained within the cleft formed by the two α-ß domains. These two domains then wrap around the ligand. This involves only minor conformational changes; the ligand-binding site is fairly inflexible. It should be noted that the binding of bicarbonate is coupled to the binding of Ca2+. The two ions bind co-operatively with high affinity. It is thought that Ca2+ may act as a cofactor.
 
  
 
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Revision as of 20:32, 13 October 2016

Click on different parts on the picture to find out more!

CmpA Gene

Overview

CmpA encodes the substrate-binding protein of the HCO3- transporter and plays a crucial role in the carbon-concentrating machinery of synechocystis, necessary for efficient energy production via photosynthesis. CmpA has never been made into a Biobrick, and the CLSB UK team synthesized and then characterized this part in our model organism. Despite it having the highest affinity for Bicarbonate and Carbonic acid in the Synechocystis genome it is the rate limiting step of CO2 absorption in the CmpAD operon, so in theory to overexpress it would increase the rate of growth of Synechocystis, especially important as the current slow rate of growth is one of the largest issues with using Synechocystis as a model organism.

The Operon:

CmpA forms the first part of the CmpABCD operon, coding for proteins that allow dissolved Carbon Dioxide to be transported into cyanobacteria for use in photosynthesis. CmpA is the substrate binding membrane protein, CmpB is an integral channel protein, and CmpC and D are ATPases to drive the active transport, as well as CmpC controlling the transport rate.

The operon is part of the CO2 concentrating mechanism (CCM) that allows CO2 to be concentrated up to 1000 times around Rubisco, an essential enzyme which catalyses a reaction with CO2 in photosynthesis and is inhibited by oxygen. Since the Great Oxidation Event, the inhibition of Rubisco by Oxygen caused Rubisco to be very inefficient, and the concentration of CO2 in the Carboxysome results in a significant increase in photosynthetic efficiency. As cyanobacteria are water dwelling, CO2 is transported in its dissolved form – H2CO3 or HCO3- depending on the pH. This is converted to CO2 in the carboxysome.

CmpA has the highest affinity to both the bicarbonate ion and carbonic acid of any protein in the cyanobacteria genome. It is used to transport bicarbonate ions (HCO3- ) into the CmpB channel.

Figure 1. Structure of CmpA-D in the membrane

Structure

CmpA is an α-ß protein similar in structure to the nitrate transporter NrtA. It consists of two domains arranged in a C-clamp shape around the ligand-binding site. Both of these domains contain a central complex of five-stranded ß-pleated sheets surrounded by α-helices. They are joined to each other by a sheaf of irregularly coiled elements. The entire protein is anchored to the plasma membrane by a lipid; CmpA is a member of the periplasmic-binding protein superfamily.

Mechanism

Bicarbonate ions bind to the ligand-binding site contained within the cleft formed by the two α-ß domains. These two domains then wrap around the ligand. This involves only minor conformational changes; the ligand-binding site is fairly inflexible. Above is a ribbon diagram showing CmpA at pH 5.0 on the left and 8.0 on the right. The bicarbonate ion is shown on the left in the entrance to the cleft as a sphere. The carbonic acid is shown on the right deep inside the cleft as a sphere. Carbonic acid can only bind in the presence of Ca2+ ion, which may act as a cofactor and is also shown as a sphere on the right.

Genetic Approach

Given the availability of this gene's sequence, the most sensible approach was to design the primers for amplifying this specific DNA directly out of the Synechocystis PCC sp. 6803 genome. So, initially, we set about designing the pair of primers that would allow us to amplify this specific gene. Unfortunately, having looked at different options for this, we encountered a significant problem. The primers that would have given us the most useful stretch of DNA would have, most likely, formed primer dimers. As a result, we have opted for having the gene synthesized as a gBlock by IDT. This approach turned out to be much better as we could synthesise the gBlock that contained the RBS and BioBrick prefix and suffix, simplifying our cloning strategy. We chose to use the RBS that was described in Wang et al. 2012. We then amplified this construct using the oligo primers that we also had synthesised by IDT.

References

Koropatkin NM, Koppenaal DW, Pakrasi HB, Smith TJ. 2007. The structure of a cyanobacterial bicarbonate transport protein, CmpA. J. Biol. Chem. 282:2606–2614

Raven, J.A., Cockell, C.S., De La Rocha, C.L., 2008. The evolution of inorganic carbon 69 concentrating mechanisms in photosynthesis. Philos. Trans. R. Soc. B: Biol. Sci. 363, 2641-2650

Maeda S, Price GD, Badger MR, Enomoto C, Omata T. 2000. Bicarbonate binding activity of the CmpA protein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in active transport of bicarbonate. J. Biol. Chem. 275:20551–20555