The first protein to be sequenced - Sanger sequencing of insulin
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How do you spell insulin?
How do you spell insulin? Hint: it starts with a G and an F! Frederick Sanger - who, with colleagues in the early 1950s, correctly spelled insulin (and developed crucial protein-sequencing techniques along the way). ⠀
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full text & linksbit.ly/insulinsequencing ⠀
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Turned out the choice of protein was fortuitous in one sense - it turned out to be really small - but unlucky in some other ways - the mature form of insulin is made up of 2 chains (the A chain & B chain) that are connected through interchain disulfide crosslinks (more explanation forthcoming). So even though insulin is small (only 51 letters instead of the several hundreds many other proteins have) It took them several years to finish the job, with different pieces of the puzzle getting published along the way. The basic history is:⠀
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1945: figure out the “starting ends” ⠀
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1949: figure out the beginnings (first few letters)⠀
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1951: complete sequencing of the 30-amino-acid-long, “phenylalanyl chain” (B chain)⠀
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1953: complete sequencing of the 21-amino-acid-long “glycyl chain” (A chain)⠀
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1955: figure out the crosslinks between the chains⠀
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1958: win first Nobel Prize⠀
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1980: win second Nobel Prize (for nucleic acid sequencing)⠀
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Each step came with its own challenges and required Sanger and his colleagues to be innovative (and very patient!) The general scheme was to use different methods (e.g. partial acid hydrolysis & enzymatic digestion) to break up the chains into smaller, overlapping, peptide pieces - separate and isolate those pieces based on charge, solubility, etc. Figure out what their first letter was, and then break them up into individual letters using complete acid hydrolysis. Then separate those letters & compare the letters in the peptides to one another to determine where they overlapped and in that way piece together the whole sequence. ⠀
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When you count you begin with 1, 2, 3, when you sequence insulin you begin with DNP! Actually you start with FDNB. FDNB stands for 1, Fluoro-2,4-DiNitroBenzene. It makes it easier to absorb visible light, and it changes its solubility so that it becomes more soluble in “organic” solvents like ether. So, if you attach the benzene derivative DNPB to something (like an amino acid), you get DNP-something which will make it appear yellow & allow you to “extract” it into things like ether that non-derived amino acids avoid.
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This would give Sanger a way to label peptides & amino acids and visually track them with “real” chromatography (more on this in a minute). But how to convince the DNB to stick on? In addition to those 2 nitro groups, FDNB has a fluorine atom. And that fluorine is relatively easy to convince to leave, so FDNB can swap out the F for another nitrogen (leaving as hydrofluoric acid, HF). And it can find such a nitrogen in the amino groups of proteins. It can only do this at “end” amino groups - like the free N-termini of proteins, or the end amino group in lysine’s side chain - so it will only attach to a protein at the N-termini & lysines.⠀
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So if you label a protein with FDNB and then use acid to split the protein up into individual letters and separate those letters, you can isolate the yellow ones and, based on how quickly they travel through various solvents compared to known standards (synthesized versions of the DNB derivatives of those letters), figure out what they are - if it’s not a lysine you’ve located an N-terminal residue. If it is a lysine, it could be anywhere in the protein, but if it’s a “middle” one it’ll only have a single DNP (and different chromatographic properties). ⠀
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When Sanger did this, he isolated DNP derivatives of glycine (Gly, G) & phenylalanine (Phe, F) & a “middle” lysine. This told him he had 2 peptide chains. Now Sanger had to figure out what letters were in the chains and in what order.
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He started by using partial acid hydrolysis, which “randomly” cleaves peptide bonds. This would give him a random assortment of pieces, good for generating overlapping peptides. But he had to time things carefully - if he let it go too long he’d get pieces that were too short to be of any help - but if he didn’t let it go long enough he’d get pieces that were too long to have much usefulness since he could only figure out which letter was first (by DNP labeling) and what other letters were in the pieces, not what order they were in. ⠀
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How’d he do that? Lots of separating in different ways including fractionation via “extraction” where some peptides dissolve in one solvent while others dissolve in another solvent, so you give them a choice (mix both solvents) to kinda bulk separate them.
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This winnowed down the number of overlapping spots or bands he’d end up with when he used his more sensitive methods - a combination of electrophoretic and chromatographic techniques to separate them based on things like charge (in the case of the electrophoresis), size, & solubility in various solvents.
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در تاریخ 1400/08/12 منتشر شده
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