New
Discovery in ATP Synthase
Background Information
Role
of ATP Sythase in the Cell
Methods
Two common methods were used in this experiment: photo-cross-linking and MALDI mass spec.
Photo-cross-linking is a method that can be used to study protein-lipid interactions (as well as interactions of proteins with other molecules). This method involves the attachment of photoactivatable probes to cysteine residues added to the protein sequence. When the probe is exposed to a certain wavelength of light, it forms a covalent linkage with molecules around it. In this experiment, several E coli mutants were created by adding single cysteine residues into the c subunit at positions 4, 8 and 11 to create three separate mutants with cysteine residues in these locations. Cysteine residues were used because they are easy to alkylate with a reagent that has a photoactivatable marker on it. When this marker is exposed to light, it forms a covalent bond with molecules near it. Below are some photocrosslinking reagents used in this experiment (Oberfeld et al., 2006).
Figure : Four photocrosslinking reagents were used in this experiment: Dia-18, Dia-19, MBP and BM.
Mass spectrometry was used in this experiment to determine the mass of different cross-link products. MALDI mass spec(matrix assisted laser desorption ionization mass spectrometry) is a way of determining the mass of a molecule by ionizing it and then determining its mass based on the charge to mass ratio. This can be determined by separating ions in a charged environment. The distance an ion travels in a charged environment is related to its mass, so this relationship can be used to determine the mass of a molecule.
Products of the photocrosslinking experiment were submitted to mass-spectrophotometric analysis to determine the mass of the products. Phospholipase C was used to diegest the cross-link product, and the difference between the purified cross-link product and the product digested with phospholipase C corresponds with the mass of phosphatidylethanolamine. The most abundant product had a mass 719 Da, 126 Da greater than that of a c subunit alone. This corresponds to the mass of phosphatidylethanolamine, therefore this increase in mass has been attributed to the presence of phosphatidylethanolamine in the interior of the c ring (Oberfeld, et al. 2006).
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| Figure : MALDI mass spec of subunit c (modified with cysteine residue, and cross-link products, purified from E. coli. Products were treated with Dia-18, which targets -SH groups (A and B) but only B was exposed to light. Samples were also treated with phospholipase C. Both spectra are of ATP Synthase modified at residue 8. | |
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| Figure : Purified ATP Synthase modified at position 4 was exposed to Dia-19 (C and D), an alternate photoactivatable reagent. C shows the first fraction of products that were eluted in the purification process, and D shows a later fraction. Peak I in D is the unmodified ATP Synthase, while peak II in C is subunit C bound with cross-linking reagent Dia-18. | |
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| Figure : Purified ATP Synthase treated with MBP and expossed to light (E). The peak labeled II is the primary product of interest in the study. In the second part of the figure, unmodified ATP Synthase was purified and treated with Dia-18 as a control. The major peak corresponds to the mass of ATP Synthase. | |
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| Figure : In G, the purified ATP Synthase was not treated with any crosslinker, but was exposed to light. This serves as a control. The resulting peak appears to be mostly one product, and since no cross-link product is present, this peak represents the ATP Synthase on its own. In H, the purified ATP Synthase was exposed to BM, another cross-link reagent, but it was not exposed to light. | |
In the figures above, each peak is labeled according to the following labels: I is unmodified subunit c; II, subunit c modified with cross-linking reagent; III, subunit c modified with cross-linking reagent with sinapinic acid (matrix peak); IV, subunit c bound to phospholipid; V, subunit c modified with SH-directed photoactivatable reagent cross-linked to phospholipid; VI, unmodified subunit c with sinapinic acid (matrix peak); VII, subunit c bound to diacylglycerol; VIII, subunit c modified with SH-directed photoactivatable reagent cross-linked to diacylglycerol; IX, unidentified peak.
SDS-PAGE is a method used to compare the mass of purified proteins in a qualitative manner. SDS (sodium dodecylsulfate) is used to denature proteins in the gel. PAGE stands for polyacrylamide gel electrophoresis. This means that proteins are placed in a gel made of a material called polyacrylamide, and then a current is run through the gel. The proteins are then separated based on size: larger proteins travel shorter distances, as they can not travel through the gel as well as smaller proteins.
Figure
: In the gel labeled
A, the first lane contains wild-type subunit c, lane 2 contains subunit
c with a cysteine residue at position 8, and lane 3 shows the existence
of multimers of subunit c. The masses (in kDa) are indicated on the left
of the gel. In the gel labeled B, subunit a was labeled. Some previous
studies suggested that subunit a was found within the c ring, but no cross-link
products formed between c and a, offering evidence that counters this
claim.
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