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53 SEQUENCING The __________________ of a protein is defined as the sequence (order) of amino acids in the protein chain from the N-terminal to the C-terminal. Sequence ultimately determines the function. Sickle-cell anemia - caused by a single amino acid change from that of normal hemoglobin. _______ of proteins are _________________ - have _______________________ in sequence within a population. There are many normal human hemoglobins that vary in sequence but the functioning of the protein is not affected. Identifying the N-terminal Can __________________________________ for detection - _________________________: highly fluorescent _________________________: colored _____________________________ (FDNB) (Sanger) _____________________________ (PITC): used in Edman degradation (sequencing) 54 General scheme for sequencing of chains _____ amino acids long 1. _________________ the N-terminal amino acid ___________ 2. _______ the derivatized N-terminal amino acid from the chain. 3. Physically separate the derivatized N-terminal amino acid from the rest of the chain by _____________________. 4. Use a detection method to _______________________ of the N-terminal amino acid. _____________ is often used 5. _______________ with the rest of the chain from step 1 until you reach the last amino acid. See figure 3-25 Modifications for ______________________________ 1. Break all __________________________. 2. Cut the chain into ____________ amino acids with an enzyme. 3. Separate the pieces and _____________ each piece separately. 4. ________steps 1,2 and 3 but using a ______________in step 2. 5. Compare results to determine how pieces fit together. 6. Use _______________to determine location of disulfide bonds 55 ________________________________ 1. React with _______(phenylisothiocyantate) under mild alkaline conditions - form the PTC adduct (phenylthiocarbamyl derivative) 2. Add ___________ (lacking water)_______(trifluoroacetic acid) - (analinothiazolinanone derivative) - leaves the rest of the chain with 1 less amino acid - _______________________________ - reaction does not degrade the rest of the chain 3. Selectively extract the anilinothiozolinone derivative into an organic solvent - physically separate the layers 4. Add ___________ to get the final PTH (phenylthiohydantoin) derivative (more stable) 5. Identify the amino acid – PTH derivative by TLC, HPCL, GLC, mass spectroscopy, etc. 6. Use the remaining chain and repeat from step 1 in cycles. 56 For Chains Larger than 50 Amino Acids 1. Cleave the chain into pieces < 50 residues ________________– enzymes that ___________________ - usually a protein - cleaves at the N or C terminal of specific residues (don’t memorize, just be able to use this table) Protease Cleavage site Trypsin Lys, Arg © Submaxillarus protease Arg (C) Chymotrypsin Phe, Trp, Tyr © Staphylococcus aureus V8 protease Asp-N-protease Asp, Glu (C) Pepsin Phe, Trp, Tyr (N) Cyanogen Bromide (BrCN) Met (C) Asp, Gly (N) conditions need to be adjusted to maximize protease activity some are really more specific than others there are more besides the list above.............. 57 2. Break the disulfide bonds Consider this oligopeptide that has a disulfide bond between the cysteines: Gly-Ala-Cys-Met-Val-Phe-Cys-Gly-Gly S-S What happens when you incubate with cyanogens bromide? Gly-Ala-Cys-Met Val-Phe-Cys-Gly-Gly S-S But the two fragments are still joined by the -S-S- bond and _______________________________________. Disulfide bonds can be cleaved in several ways: _______________ by ____________________(HCOOOH) Replaces S-S with 2 SO3- 58 Reduction with ____________ (____ or _______ reagent) or with ________________________ (__-mercaptoethanol) a. both cleave the -S-S- bond to give 2 -SH groups DTT 2-mercaptoethanol b. Disulfide bonds can spontaneously oxidize by _______. Prevent by treating with ______________ (ICH3COO) This________________________ the -SH groups. 59 3. ___________________ by electrophoresis or chromatography. 4. _________________ each fragment by the Edman degradation. 5. ___________ at least one more time from the beginning with a fresh sample of protein, but choose a ____________________. Need this to put fragments in the proper order. 6. To finish the complete sequence, the location of S-S bonds must be determined. use ______________________________________ - start with 2 fresh aliquots (portions) of protein - treat one with a _________________ - treat other with the __________________ plus ______ - run samples side-by-side on a gel - If no difference between samples _________ present or only –S-S- bonds between cysteines ___________________. - If there is a difference –S-S- bonds are present. Example: 60 Importance of sequence determination ________________ to known sequences(databases) – is it new? Compare to ________________________________ (taxonomy) compare to proteins of ________________________ (__________________________ proteins) - ____________ – homologous proteins in the ___________ -_____________ – homologous proteins from _____________ - find _________________ – for example the active site of an enzyme – often highly similar from species to species - 3 types of residues 1. _________________ – identical among homologs 2. _________________ – different among homologs 3. ________________ – some difference among homologs, but substitutions are always with similar residues (e.g. _____ <-> ______ or _____ <-> ______ ) Example: cytochrome C (mitochondrial protein) 100 residues MW ~ 13,000 comparing 60 species 27 invariant 61 Computer programs used to compare sequences and determine relatedness and/or evolutionary history. Can weight importance of substitutions with the ________ table (blocks substitution matrix). Identify _________________________ – unique for the group in which they are found (species, genus, etc.) Synthesis of peptides: Automated (so is sequencing) Critical component is the _____________ at each reaction step. Can synthesize a ______ amino long polypeptide in few days. A cell can do this in 5 sec! 1. Attach amino acid to a column resin. ______________ protects amino end from reaction 2. Remove Fmoc 3. Protect (Fmoc) and ______ (dicyclohexylcarbodiimide, DCC) the next amino acid 4. Attach 2nd amino acid to one on the column 5. Repeat until peptide as long as desired 6. Use ______ to deprotect final amino acid and remove chain from the column. 62 63