00Note Set 5b 1 PEPTIDE BONDS AND POLYPEPTIDES OLIGOPEPTIDE: --chain containing only a few amino acids (see tetrapaptide, Fig 5.9) POLYPEPTIDE CHAINS: --many amino acids joined together --not necessarily a protein! (all proteins are polypeptides, but the converse is not true) a protein has a specific amino acid sequence that is defined by a gene amino acids in a polypeptide are called amino acid residues polypeptides (and proteins) have a front end (amino terminus or N-terminus) and a back end (carboxyl terminus or C-terminus) most proteins contain 50-2000 amino acids mean molecular weight of an amino acid is 110 (see Problem #1) so MW of proteins could be 5500 to220,000 (ball park numbers) Polypeptides as polyampholytes (substance whose groups have both acidic and basic groups) they have many weakly acidic and basic groups (Fig 5.11) even a small shift in ph can significantly affect the structure and interactions of a protein molecule Molecular weight does not have units mass has units called daltons
00Note Set 5b 2 mass of above proteins would be 5500 daltons to 220,000 daltons (how many kd?) PEPTIDE BONDS: amide bond between α-amino and α-carboxyl groups structure solved by Pauling and Corey No rotation around peptide bond...a rigid, planer unit (Fig 5.12) so the -C=O and -N-H bonds are nearly parallel (O, C, N, H usually coplanar) 1. x-ray crystallographic studies of synthetic peptides indicated that distance of C-N bond was shorter that would be expected if it had only single bond character, so must have double bond properties 2. rotations are permitted only around the α carbon 3. depending on the R group, a constraint can be imposed on these rotations by steric hindrance 4. Therefore, trans form usually favored (-X-Pro can be cis althought trans still favored; prolyl cis-trans isomerases an important new class of regulatory enzymes STABILITY AND FORMATION OF THE PEPTIDE BOND formed by dehydration (loss of H2O) (Fig 5.8) requires input of free energy, about +10 kj/mol (hydrolysis favored but very slow w/o catalyst, so peptides are stable, similar to nucleic acids) can be hydrolyzed in hot 6N HCl (see below) or by proteolytic enzymes or proteases (see Table 5.4) that often cleave at specific residues see CNBr reaction, Fig 5.13
00Note Set 5b 3 since synthesis unfavorable, like with nucleic acids, amino acids are activated by an ATP-driven reaction before incorporation into proteins = coupling each amino acid to the 3 end of its trna to yield the aminoacyl-trna, catalyzed by aminoacyl-trna synthetases, and ATP is hydrolyzed to AMP (Fig 5.19) PRIMARY STRUCTURE, proteins of defined sequence Primary structure is the specific sequence of amino acids occuring in a protein defined by genes Fred Sanger sequenced the first protein in 1953: bovine insulin 1st Nobel, because it showed that: the sequence is precisely defined only L-amino acids are found they are linked by peptide bonds thousands of proteins have now been sequenced each is unique Why is the primary structure important? helpful in elucidating mechanism of action determines the three dimensional structure, which confers biological function rules that govern protein folding are being discovered by studying the relationship between primary structure and three dimensional structure very important to an emerging area of medicine: molecular pathology one amino acid change in the primary structure can lead to abnormal function and disease
00Note Set 5b 4 examples: sickle cell anemia and cancer CHARACTERIZATION OF PRIMARY STRUCTURE Examples of primary structure: Mb myoglobin (3D Fig 5.1, primary Fig 5.14) and see Table 5A.1 for how to isolate and purify Mb) Insulin (Fig 5.15 and 5.21) Insulin has disulfide bonds: disulfide bonds cross-links between chains or within a chain formed through oxidation between pairs of cys side chains product of the oxidation called cystine PROTEIN PURIFICATION For example, from < 0.1% of starting material (total protein) to 98% pure 1. Requirements: Assay Good supply of starting material Overexpession in E coli or other organism not without problems...not as easy Patience as the book makes it sound 2. What kinds of proteins are easy to purify?
00Note Set 5b 5 Abundant proteins Proteins that have some unique property General procedures 1. Stabilization: Use buffers at appropriate ph to prevent denaturation and/or degradation Purification often done in cold because proteases less active...not so important if using HPLC which is very fast Protease inhibitors are often used to prevent degradation Minimize foaming Keep concentrated, proteins usually are more stable in a concentrated solution. Especially important for long-term storage. Usually stored at -20 C to -80 C Assays 1. Use a direct assay if available, as for an enzyme, or a coupled reaction if a detectable product is not made by a direct assay Amount of product formed is proportional to the amount of enzyme present 2. Use antibodies to detect purification, if can be made through reverse genetics Separation Techniques Homogenation Subcellular fractionation Differential centrifugation
00Note Set 5b 6 Separation on the basis of charge, polarity, size, binding specificity, temperature stability and other properties Solubility Solubilize appropriate fraction: Salt step ("salting out") Different proteins precipitate at different salt concentrations "ammonium sulfate cut" Chromatography Ion exchange DEAE cellulose (or agarose) = anion exchange, CM cellulose = cation exchange Reverse Phase = hydrophobic interaction chromatography, nonpolar alkyl groups Affinity attached to matrix Gel filtration (Fig 5A.5) Silica beads with pores HPLC Gel electrophoresis See one band on two different kinds of gels!! PAGE (Native) SDS-PAGE (Denaturing) Isoelectric Focusing
00Note Set 5b 7 Determination of amino acid compostition hydrolysis of protein (usually in 6N HCl at 110 ) hydrolysate (amino acids) is separated by ion exchange chromatography a column of beads that separates molecules on the basis of charge there are cation exchange (-) columns and anion exchange (+) columns amount of each amino acid is then quantitated by reaction with ninhydrin (Fig 5B.2) (darkness of resulting blue color is proportional to the amount of amino acid that is present); Or Fig 5B.1 use a single column amino acid analyzer, can be done by HPLC identification of amino and carboxy terminal residues amino terminal residue: rxn with dabsyl or dansyl chloride forms dabsylated or dansylated derivative which can be identified by chromatography after cleavage in 6N HCl destroys peptide so only amino term. amino acid can be identified carboxy terminal residue: rxn with carboxypeptidase A removes carboxy terminal residue composition can then be redetermined to see what's missing Cut up the protein and sequence the pieces... cut up the protein:
00Note Set 5b 8 all cut after the indicated amino acid(s) trypsin: lys or arg clostripain: arg chymotrypsin: phe, trp, tyr thermolysin: leu, ile, val CNBr: met more in your book Sequence the pieces: Edman degradation (Fig 5C.1) done these days by machine react with PITH and cleave does not detroy protein so can do over and over analyze PTH-derivative also analyze what's left see example of sequencing of the B chain of insulin in Fig 5C.2 and locating the MS-MS disulfide bonds in Fig 5C.4 tandem mass spectroscopy inject peptides into first sector vaporize in vacuum by FAB or electrospray
00Note Set 5b 9 moves into second sector smashed into random, different smaller pieces by collisions with inert gas pieces move to detector via a strong magnetic field which allows the determination of the mass of each piece analyze data and put puzzle together