Determination of Primary Structure

OUTLINE I. Protein Structure A.Primary 1.Determination a. Sequence determination; CHEMICAL i. ii. iii. iv. v. Cleavage of peptides bonds Amino acid composition and stoichiometry Disrupt and determine number of chains; Divide & Conquer; Edman Degradation b. Sequence determination; PHYSICAL i. Mass Spectrometry for proteins ii. Use of tandem MS/MS for sequence determination iii. Isolation of proteins by 2D PAGE; Isoelectric focusing x SDS-PAGE c. Sequence determination; BIOLOGICAL i. Genome sequenced ii. Bioinformatics to predict protein sequences in predicted genes iii. Use of CHEMICAL and/or PHYSICAL methods to get partial sequence Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 1 Determination of primary structure Amino Acid Composition Absorbance is at 570 nm (purple). How did they make the amino acid residues colored? …......ninhydrin How many AA are “resolved?” …....18 Where are the other 2? …....Asn & Gln have already been converted to Asp & Glu, respectively Determination of primary structure Amino Acid Composition Cation exchanger=Dowex-50 (sulfonic acid on polystyrene) Calculate the “mole%” or “mole fraction”; in other words the stoichiometry: Example; protein MW = 11,000 Da Approximate #AA = 11,000/110 (Ave MW for AA) = 100 AA If 10% of A570 is 10% for Arg: 100 x 0.10 = 10 Arg in protein 2 Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 38 Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 3 Disrupt structure (2°, 3°, 4°, and disulfides) What holds these levels of structure together? …....non-covalent bonds (H-bonds, van der Waals, ionic, hydrophobic) What have you used in the lab that might disrupt non-covalent bonds? …....Urea, SDS, pH extremes, heat, etc. What about the covalent S-S bond? …....2-mercaptoethanol (b-mercaptoethanol, BME) or dithothreitol (DTT). To keep disulfides from reforming….. 1) keep BME at high concentration in buffers 2) alkylate or oxidize the SH groups Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 4 Determine the number of peptide chains by counting number of amino terminal ends Example: • • • • Frederick Sanger (1918-2013) small tripeptide AA comp = A,K,L C-term = K* DNP-A Sequence is A-L-K Sanger’s Reagent Absorbs at 353 nm (yellow) *Used carboxylpeptidase (more later) Determine the number of peptide chains by counting number of amino terminal ends Dansylation Fluorescence detection is hundreds of times more sensitive: less protein needed F519 nm Excitation at 330 nm Emission at 519 nm 5 Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 6 Divide into fragments: Proteolytic Cleavage “TABLE 5-5” Specificities of Exopeptidase O– Carboxylpeptidase A Bovine pancreas Rn = C-terminal; Rn-1 ≠ Pro Divide into fragments: Cyanogen Bromide Cleavage Methionine Imine intermediate (Schiff base) Similar result as an endopeptidase, but leaves a homoserine lactone as the C-terminal residue 7 Separation and isolation of peptide fragments Example: paper electrophoresis Also, TLC, silica gel, etc. to Ninhydrin 48 Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends, by Dansyl chloride or FDNB 5) & 6) Divide into fragments, using 2 different proteases that cleave peptide bonds only after specific residue, and determine sequence with Edman degradation. 7) Determine overlaps and piece original sequence back together 8 Determination of primary structure: Divide & Conquer 51 Determination of primary structure: Divide & Conquer 52 9 Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 53 Determine the Sequence: Edman degradation Phenylthiocarbamyl (PTC)-polypeptide Edman Degradation Animated Figure (https://wileyassets.s3.amazonaws.com/Voet_Fundamentals_of_Biochemistry_5e_ ISBNEPROF12533/media/animated_figures/ch05/5-15.html) Phenylthiohydantoin (PTH)-amino acid 10 Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 55 Determine overlaps and piece original sequence back together 11 Determination of primary structure THREE basic ways to know the primary structure. Only the CHEMICAL method will give the entire covalent structure, including any disulfide bonds. But other methods are more sensitive. One can classify these methods by: CHEMICAL PHYSICAL “BIOINFORMATICAL” We just went through the CHEMICAL. The PHYSICAL method still requires the same strategy, including purification, fragmentation, chromatography, and alignment. But, instead of an Edman degradation the use of tandem Mass Spectrometry (MS) is employed. Lets look at the use of MS in biochemistry • • • • • Ions “fly” in a vacuum toward a target with a velocity ∝ z/m (charge-to-mass ratio) Molecules with higher charge and lower mass get detected first. Molecules with a lower charge and higher mass get detected last. Plotted as m/z to read peaks from left to right Instruments can distinguish molecules with same charge by < 1 Da Determine the Sequence: Tandem MS The major problem in using MS for macromolecules is getting them to “fly” in a vacuum with a charge. TWO major methods: 1) Electro-Spray Ionization (ESI) 2) Matrix-Assisted ESI LaserDesorption Ionization Mass = (m/z) x z (MALDI) (m/zA) x zA = (m/zB) x zB 893.3z = 848.7(z+1) 893.3z = 848.7z+848.7 What is 848.7 = z(893.3–848.7) = z44.6 MALDI? 848.7/44.6=19 = z Mass = 893.3x19 = 16,973 Mass = 848.7x20 = 16,974 Mass = 1696.3x10 = 16,963 Figure 5-17 12 Determine the Sequence: Tandem MS Matrix-Assisted LaserDesorption Ionization (MALDI) MALDI How do you use MS to get protein sequences? Determine the Sequence: Tandem MS • Mass spectrometry uses mass-to-charge ratio of different ions to determine mass • Tandem MS-MS: First selects a peptide, then fragmentation, and second determines mass of fragments • By comparing results of all fragments, you can find masses that differ by mass of one amino acid to determine sequence Nobel Prize in Chemistry 2002 13 Determine the Sequence: Tandem MS • EXAMPLE AVAW (m=495) AA MW Ala 90 Val 109 Trp 206 c3 c2 C1(y1) +NH 3 CH3 a3 CH CH3 CH3 CH3 CH a2 a1(b1) AVAW 199 90 289 296 405 495 206 a3 a2 c1 a1 c2 c3 m0 Determine the Sequence: Tandem MS • EXAMPLE AVAW AA MW Ala 90 Val 109 Trp 206 Another method first selects for just the Cc2 peptides C1(y1) c3 +NH 3 CH3 a3 CH CH3 CH3 CH3 CH a2 a1(b1) AWVA 199 296 AVAW 405 495 90 90 a3 c2 c1 199 a2 a1 c3 m0 289 296 405 495 206 a3 a2 c1 a1 c2 c3 m0 There is an issue with K & Q, which have the same MW! 14 Determine the Sequence: Tandem MS • EXAMPLE AVAW AA MW Ala 90 Val 109 Trp 206 c3 c2 C1(y1) +NH 3 CH3 a3 CH CH3 CH3 CH3 CH a2 a1(b1) AWVA 199 296 AVAW 405 495 199 90 90 a3 c2 c1 a2 c3 a1 m0 289 296 405 495 206 a3 a2 c1 a1 c2 c3 m0 There is an issue with K & Q, which have the same MW! Determination of primary structure THREE basic ways to know the primary structure: Edman Degradation requires >100 pmole (1-5 µg) CHEMICAL MS/MS requires >1-10 pmole (100-500 ng) PHYSICAL BIOINFOMATICAL We just went through the CHEMICAL and PHYSICAL. The BIOINFORMATICAL method requires information from chemical or physical, but only a limited amount of sequence. • Example: a sequence of 6 AA is only possible as one of 206 possible hexa-peptide sequences (1 of 64x106). • There are no more than 50,000 protein-coding genes with ≤400 AA on average. This is ~20 x 106 possible unique sequences. • So, a hexamer is not likely to appear more than once. • Once you have at least 6 AA sequence, you can compare that to all possible proteins encoded in the entirety of the gene sequences (genome) for a species for which the genome is known using appropriate bioinformatic tools. This will then give you the entire protein sequence. There is one remaining issue: Where are the Disulfides, if any? …...This requires chemical and/or physical methods 15 Determination of primary structure THREE basic ways to know the primary structure: Edman Degradation requires >100 pmole (1-5 µg) CHEMICAL MS/MS requires >1-10 pmole (100-500 ng) PHYSICAL BIOINFOMATICAL We just went through the CHEMICAL and PHYSICAL. The BIOINFORMATICAL method requires information from chemical or physical, but only a limited amount of sequence. • Example: a sequence of 6 AA is only possible as one of 206 possible hexa-peptide sequences (1 of 64x106). • There are no more than 50,000 protein-coding genes with ≤400 AA on average. This is ~20 x 106 possible unique sequences. • So, a hexamer is not likely to appear more than once. • Once you have at least 6 AA sequence, you can compare that to all possible proteins encoded in the entirety of the gene sequences (genome) for a species for which the genome is known using appropriate bioinformatic tools. This will then give you the entire protein sequence. There is one remaining issue: Where are the Disulfides, if any? …...This requires chemical and/or physical methods Determine which Cys are in Disulfide bonds Cys Cys Cys Wiley Guided_explorations Cys Which of these Cys is bonded to…. This Cys Cys (https://wileyassets.s3.amazonaws.com/Voet_Fun damentals_of_Biochemistry_5e_ISBNEPROF12 533/media/guided_explorations/ge_04_Protein_s equence_determination/protein_sequence.html) Figure 5-13 16 Determine which Cys are in Disulfide bonds Recall that to separate polypeptides for sequence analysis, the disruption of –S-Sbonds is needed And, that prevention of their re-oxidation can be done by alkylation or oxidation. = O HCOOH Performic Acid Oxidize disulfide, which breaks -S–S- bond – O3 Sulfonic acids + – O3 Prevention of their re-oxidation can be done by peroxidation as well. Separate and sequence the polypeptides How do we use this to find the Disulfides? Determine which Cys are in Disulfide bonds We change the protection step for sulfhydryl groups, instead of before: • Cleave/Protect (reduction/alkylation or oxidation) AFTER fragmentation • Separate fragments as before, but any linked by –S–S- will not separate and remain together (e.g., orange peptide). • THEN break –S–S- bonds, and re-separate. Determine the sequence of those peptides that fall off the diagonal by either Edman degradation or tandem MS/MS. Technique is called ”2D-diagonal electrophoresis.” 17 Determine which Cys are in Disulfide bonds We change the protection step for sulfhydryl groups. • Cleave/Protect (reduction/alkylation or oxidation) AFTER fragmentation • Separate fragments as before, but any linked by –S–S- will not separate and remain together (e.g., orange peptide). • THEN break –S–S- bonds, and re-separate. Wiley Guided_explorations Determine the sequence of those peptides that fall off the diagonal by either Edman degradation or tandem MS/MS. Technique is called ”2D-diagonal electrophoresis.” Determine which Cys are in Disulfide bonds We change the protection step for sulfhydryl groups. • Cleave/Protect (reduction/alkylation or oxidation) AFTER fragmentation • Separate fragments as before, but any linked by –S–S- will not separate and remain together (e.g., orange peptide). • THEN break –S–S- bonds, and reseparate. Determine the sequence of those peptides that fall off the diagonal by either Edman degradation or tandem MS/MS. Technique is called ”2D-diagonal electrophoresis.” Wiley Guided_explorations 18