--not necessarily a protein! (all proteins are polypeptides, but the converse is not true)

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--chain containing only a few amino acids (see tetrapaptide, Fig 5.9)


--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


mass of above proteins would be 5500 daltons to 220,000 daltons (how many kD?)


•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


•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


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


For example, from < 0.1% of starting material (total protein) to 98% pure 1. Requirements:


Good supply of starting material

Overexpession in E coli or other organism not without problems...not as easy as the book makes it sound



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


Subcellular fractionation Differential centrifugation


Separation on the basis of charge, polarity, size, binding specificity, temperature stability and other properties


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

attached to matrix Affinity

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


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...


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 disulfide bonds in Fig 5C.4


tandem mass spectroscopy inject peptides into first sector


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





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