lecture6note-yeo11 - Proteins Primary structure Chapter 5...

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Unformatted text preview: Proteins: Primary structure Chapter 5 (2/3/2011) • Polypeptide diversity • Protein Purification (later) • Protein sequencing – Preliminary steps – Polypeptide cleavage – Edman degradation – Mass Spectrometry – Reconstituting the protein’s sequence • Protein evolution Protein function as: 1. Enzymes:biological catalysts 2. Regulators of catalysis - hormones 3. Transport and store: O2, metal ions, sugars, lipids, etc. 4. Contractile assemblies: Muscle fibers Separation of chromosomes 5. Sensory: Rhodopsin nerve proteins 6. Cellular defense immuoglobulins Antibodies Killer T cell Receptors 7. Structural Collagen Silk, etc. Function is dictated by protein structure!! There are four levels of protein structure 1. Primary structure 1° = Amino acid sequence of the peptide chain(s), the linear order of AA’s. Remember from the N-terminus to the C-terminus Above all else this dictates the structure and function of the protein. 2. Secondary structure 2° = Local spatial alignment of amino acids without regard to side chains. Usually repeated structures Examples: -helix, -sheets, random coil, or -turns 3. Tertiary Structure 3° = the 3 dimensional structure of an entire peptide. (e.g. folding of secondary structural elements against one another). Great in detail but vague to generalize. Can reveal the detailed chemical mechanisms of an enzyme. 4. Quaternary Structure 4° = two or more peptide chains associated with a protein. Spatial arrangements of subunits. Example of each level of protein structure Other components of proteins • In addition to the 20 standard amino acids, some proteins have something more, such as – Metal ions (Zn2+, Mg2+, Ca 2+, etc.) – Co-enzymes, such as pyridoxal phosphate (vitamin B6 derivative), flavins, NADH, NADPH, etc. – Post-translational modifications, such as phosphorylation, addition of carbohydrates, carbamylation, etc. – Lots of water • These cofactors and coenzymes play both structural and functional (i.e. catalytic) roles Protein Sequencing Insulin was the first protein to be sequenced F. Sanger won the Nobel prize for protein sequencing. It took 10 years, many people, and it took 100 g of protein! Today it takes one person several days to sequence the same insulin and requires only a few micrograms of the protein. Importance of protein sequences 1. Knowledge of the sequence is a pre-requisite to determining protein structure 2. Comparisons among sequences can help to deduce function and evolutionary relationships between proteins in the same or different organisms 3. A large number of diseases are caused by a small number (1-2, sometimes more) changes (mistakes) in a protein sequence. Sequencing of the DNA (from which protein sequence arises) of relevant genes can help in the development of “genetically-based” drugs. Chapter 5.3 is how to determine a protein’s primary structure. “Protein Chemistry” Steps towards protein sequencing Above all else, purify it first!! Chapter 5.3 then 5.1 and 5.2 (later!) 1. Prepare protein for sequencing a. Determine number of chemically different polypeptides. b. Cleave the protein’s disulfide bonds. c. Separate and purify each subunit. d. Determine amino acid composition for each peptide. 2. Sequencing the peptide chains: a. Fragment subunits into smaller peptides 50 AA’s in length. b. Separate and purify the fragments c. Determine the sequence of each fragment. d. Repeat step 2 with different fragmentation system. 3. Organize the completed structure. a. Span cleavage points between sets of peptides determined by each peptide sequence. b. Elucidate disulfide bonds and modified amino acids. At best, the automated instruments can sequence about 50 amino acids in one run! Proteins must be cleaved into smaller pieces to obtain a complete sequence. Overview of Protein Sequencing 1.Prepare protein for sequencing 2. Sequencing the peptide chains 3. Organize the completed structure. End Group Analysis How many peptides in protein? Bovine insulin should give 2 N-termini and 2 C-termini N-terminal identifying agent 1-Dimethylaminonaphthalene-5-sulfonyl chloride Dansyl chloride Reacts with amines: N-terminus (primary amino group side chains) Detected by its intense yellow fluorescence The dansyl chloride reaction - used for end group analysis Carboxypeptidase cleavage at the C-terminus Rn-2 O NH CH C H2 O NH Rn-1 CH O C NH Rn CH O C O Carboxypeptidase Rn-2 O NH CH C NH Rn-1 CH O C O H3 N Rn CH O C O Carboxypeptidase A Carboxypeptidase B Rn R, K, P Rn-1 P Rn= R, K Rn-1 P Aminopeptidases cleave residues from the N-terminus Carboxy- and amino-peptidases are collectively known as exopeptidases Cleavage of disulfide bonds Permits separation of polypeptide chains Prevents refolding back to native structure Performic acid oxidation Cystine (-S-S-) or cysteine (-SH) to Cysteic acid (-SO3-) Methionine to Methionine sulfone, Trp destroyed 2-Mercaptoethanol, dithiothreitol, or dithioerythritol Keeps the equilibrium toward the reduced form -S-SSee V&V&P pg 112 for structures 2SH 2S-CH2COOiodoacetate Cleavage of disulfide bonds Amino acid composition The amino acid composition of a peptide chain is determined by its complete hydrolysis followed by the quantitative analysis of the liberated amino acids. Acid hydrolysis (6 N HCl) at 120 oC for 10 to 100 h destroys Trp and partially destroys Ser, Thr, and Tyr. Also Gln and Asn yield Glu and Asp Base hydrolysis 2 to 4 N NaOH at 100 oC for 4 - 8 h. Is problematic, destroys Cys Ser, Thr, Arg but does not harm Trp. Amino acid analyzer In order to quantitate the amino acid residues after hydrolysis, each must be derivatized at about 100% efficiency to a compound that is colored. Pre- or post-column derivatization can be done. These can be separated using HPLC in an automated setup - each AA has known elution time Amino acid compositions are indicative of protein structures Leu, Ala,Gly, Ser, Val, Glu, and Ile are the most common amino acids. His, Met, Cys, and Trp are the least common. Ratios of polar to non-polar amino acids are indicative of globular or membrane proteins. Certain structural proteins are made of repeating peptide structures i.e. collagen. See Table 4-1 !! Long peptides have to be broken to shorter ones to be sequenced Disadvantage with the dansyl-chloride method is that you must use 6M HCl to cleave off the derivatized amino acid, this also cleaves all other amide bonds (residues) as well. Edman degradation with Phenylisothiocyanate, PITC • Edman degradation used to automatically sequence the <50 AA peptide fragments • Sequentially degrades inward from the N-terminus • PTH-amino acid is then identified by signature TLC, HPLC, MS, gel electrophoresis, GLC, etc. Edman degradation has been automated as a method to sequence proteins. The PTH-amino acid is soluble in solvents that the protein is not. This fact is used to separate the tagged amino acid from the remaining protein, allowing the cycle of labeling, degradation, and separation to continue. Even with the best chemistry, the reaction is about 98% efficient. After sufficient cycles more than one amino acid is identified, making the sequence determination error-prone at longer reads. Reconstructing the protein’s sequence Specific chemical cleavage reagents: Cyanogen Bromide Rn-1 = M Cleave the large protein using i.e trypsin, separate fragments and sequence all of them. (We do not know the order of the fragments!!) Cleave with a different reagent i.e. Cyanogen Bromide, separate the fragments and sequence all of them. Align the fragments with overlapping sequence to get the overall sequence. How to assemble a protein sequence? 1. Write a blank line for each amino acid in the sequence starting with the N-terminus. 2. Follow logically each clue and fill in the blanks. 3. Identify overlapping fragments and place in sequence blanks accordingly. 4. Make sure logically all your amino acids fit into the logical design of the experiment. 5. Double check your work. Cyanogen Bromide (CN Br) Cleaves after Met i.e M - X D-I-K-Q-M K K-F-A-M Y-R-G-M Trypsin cleaves after K or R (positively charged amino acids) Q-M-K G-M-D-I-K F-A-M-K Y-R 6 7 8 9 10 11 12 13 14 H3N- _-_-_-_-_-_-_-_-_-_-_-_-_-_-COO 1 2 3 4 5 K F-A-M-K K-F-A-M Q-M-K D-I-K-Q-M G-M-D-I-K Y-R-G-M Y-R ...
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