Protein Sequencing - Chapter 2 (PDF)

⦁ In this chapter we will discuss the primary st. of proteins Each amino acid unit is called residue 1- For structural studies 3 D shape that will help understanding its molecular mechanisms of action 2.Evolutionary Relationships: sequence comparisons of related proteins in/between organisms...

⦁ In this chapter we will discuss the primary st. of proteins Each amino acid unit is called residue 1- For structural studies 3 D shape that will help understanding its molecular mechanisms of action 2.Evolutionary Relationships: sequence comparisons of related proteins in/between organisms shed light upon how proteins function and the evolutionary relationships between organisms. 3. Clinical Applications: mutations in aa sequence lead to many diseases, knowing this help to develop therapies ⦁ First complete aa sequence determined by Sanger in 1953 using 100g of protein. Now we can determine the sequence of a protein using few µg of protein. ⦁ 51 aa, 2 chains: α and β chains linked by S-S bond. ⦁ S-S bonds exist in inter and intrachains. How to determine the sequence of a protein?? *Determine the nb of subunits * Clevage of the disulfide bridges *Separation and purification of each polypeptide chain(s) *Cleave each chain(s) into fragments < 50 aa (Repeated) * Separation and purification of all fragments (Repeated) Sequencing the fragments using the 'Edman reagent' (Repeated) *by comparing the overlaps of different sets of fragments *Elucidate the positions of the disulfide bonds  ⦁ The number of chains can be determined by: - SDS-PAGE - End group analysis : by identifying the number of N- and C-terminal. Identification of N-terminal: - Sanger’s method (only for di-tripeptides) or FNDP (1-Fluoro-2,4-dinitrobenzene) method - Dansyl chloride - Phenylisothiocynate (PITC)/ Edman reagent - Aminopeptidase Identification of C-terminal: - Exopeptidase - Chemical reagent: Hydrazine 1- End Group Analysis  1-A Identification of N-terminal Ex: Insulin, phe N-ter and gly-N-ter  1-A-1 Acid hydrolysis of the protein; Hydronium/water Only the labeled N-terminal amino acid can be detected by a colorimetric detection at specific wavelength  1-A-2 OH- DimethylAminoNaphthalene Polypeptide Sulfonyl chloride (acid hydrolysis) (Dansyl chloride) Nb of dansyl-aa is the nb of chains/subunits Dansylamino acid (fluorescent) Free amino acids  1-A-3 OH- Phenylthiocarbamyl (Trifluoroacetic acid) (PTC) polypeptide Alkaline condition so the amino hydrolysis group is uncharged Original protein (minus its N It is the most useful methof of N- terminal residue) terminal residue identification. Why?? ADVANTAGE: Extracted and separated - Breaks only the first peptide bond Aquous acid -The remaining peptide after the removal of the N-terminal is not hydrolyzed and can be used again to detect the next aa. Detected with UV Identified by chromatography Phenylthiohydantoin ⦁ It cleaves aa from N terminus and used to determine N Terminal aa  1-B Identification of C-terminal Exopeptidases: cleave the terminal residue Carboxypeptidase : cleaves C terminal aa Selective but can’t be used to determine sq of aa: 1. Enz remove 1 residue after another complicates detection but yields some C- bonds cleaved at the same rate terminal sequence information 2. ≠ reaction rates for ≠ residues can yield an error in the order of aa  (aromatic,branched) = Normally we must confirm C-terminus identification using several, different carboxypeptidase Treatment with hydrazine at 90◦ for 20—100 h in the presence of acidic Ion Exchange Resin (catalyst). Aminoacyl hydrazides are not eluted Many side reactions → limit its use.  B- Cleavage of disulfide bonds WHY?? 1- Separation of polypeptide 2- Open the 3-D conformation: chains Protein cleavage  Reduction of Disulfides are required to separate and purify individual polypeptide chains Denaturing conditions: heat (to expose the SH) S-S reducing agents: 2- mercaptoethanol, dithiothreitol ⦁ ALKYLATION: To prevent reformation of S-S (cystine) through oxidation by O2 Hydriodic acid Stable compound S is going to be attached to carboxymethyl group 1. Denaturation: acidic-basic- low salt- high temperature- denaturant (urea- guanidin hypochloride- SDS) 2. Separation of polypeptide chains on the basis of charge (polarity) IEC or size by SEC-HPLC 3. Estimate the nb of residues from the MW( 110Da/residue) By SEC-SDS- PAGE, MS (fast, accurate, even for picomolar) ⦁ Direct sequencing is applicable to 40-60 aa ⦁ Problems occur after lengthy reactions - Incomplete reactions - Accumulation of impurities from side reactions ⦁ Solution: - Use enzymes (endopeptidases) or chemicals (highly specific) (CNBr) to fragment the polypeptide chain. Endopeptidases (cleave internal peptide bonds) have side chain requirements for the aa near the scissile (to be cleaved) peptide bond)  Broader specificities and yield peptides with overlapping sq. Adjusting reaction times and conditions limits the number of sites cleaved (ie. Limited proteolysis). Can optimize fragmentation conditions and maximize number of fragments of useful size.  CH3 Reaction performed in acidic solvent: HCL or 70% formic acid In acidic solvent (HCOOH or HCl) + to denatures most proteins so that cleavage occurs at all Met HPLC  Compare amino acid sequence of one set of peptide fragments with an overlapping second set of fragments obtained using different cleavage points. CNBr fragments Trypsin fragments Phe-Trp-Met Phe-Trp-Met-Gly-Ala-Lys Gly-Ala-Lys-Leu-Pro- Met Leu-Pro- Met-Asp-Gly-Arg Asp-Gly-Arg-Cys-Ala-Gln Cys-Ala-Gln Combine information from two sets of fragments into complete sequence Phe-Trp-Met-Gly-Ala-Lys-Leu-Pro-Met-Asp-Gly-Arg-Cys-Ala-Gln  Cleaving polypeptide chain(s) while keeping the S-S bond intact Repeat fragmentation reaction Separate the peptide fragments by reverse phase HPLC Compare protein samples without S-S with that of intact S-S=> The S-S linked fragments are identified and subjected to Edman’s degradation