Amino Acids, Peptides and Proteins

Amino Acids, Peptides and Proteins • The Amino Acids in Proteins • Polypeptides and Proteins • Protein Function • Protein Size, Composition and Properties • Four Levels of Protein Structure • Protein Primary Structure • Chromatography and Electrophoresis of Proteins

Amino acids • All proteins are composed of amino acids. • Twenty common amino acids. • All are -amino acids except proline. • A primary amine is attached to the  carbon - the carbon just after the acid group.  carbon H | R-C-COOH | NH2 General Structure

Amino acids • Because an acid and base are both present, an amino acid can form a +/- ion. • H H • | | • R-C-COOH R-C-COO- • | | • NH2 NH3+ • How well it happens is based on pH and the type of amino acid. Called azwitterion.

-Amino acids • Except for glycine, the  carbon is attached to four different groups - chiral center. • Carbohydrates • We use the D-form. • Amino Acids • We use the L-form. COO- | H3N - C - H | R +

Classification of amino acids • The -amino acid group is the same in each. • Classified by the type of side chain. • Group I. non-polar side chains. • Group II. polar, uncharged side chains • Group III. acidic side chains • Group IV. basic side chains

Group I. Non-polar side chains H3CH \ | HC-C-COO- / | H3C+NH3 H | CH3-C-COO- | +NH3 alanine valine H3C H \ | HC-CH2-C-COO- / | H3C+NH3 H3CH || H3C-CH2-CH-C-COO- | +NH3 leucine isoleucine

Group II. Polar side chains H | HS-CH2-C-COO- | +NH3 cysteine O H | || H2N-C-CH2-CH2-C-COO- | +NH3 O H | || H2N-C-CH2-C-COO- | +NH3 glutamine asparagine

Group III. Acidic side chains • Based on having a pH of 7. O H | || -O-C-CH2-CH2-C-COO- | +NH3 glutamic acid O H | || -O-C-CH2-C-COO- | +NH3 aspartic acid

Group IV. Basic side chains • Based on a pH of 7. H +| H3N-CH2-CH2-CH2-CH2-C-COO- | +NH3 lysine +NH2 H | || H2N-C-N-CH2-CH2-CH2-C-COO- | +NH3 H arginine H | CH2-C-COO- | +NH3 histidine H N H N H +

H | H2NCCOOH | R H | H2NCCOOH | R’ H O | || H2N - C - C - | R H | N - C - COOH | | H R’ + Polypeptides and proteins • Proteins are polymers made up of amino acids. • Peptide bond - how the amino acids are • linked together to make • a protein. + H2O

ala arg asn asp cys gln glu gly his ile leu lys met phe thr pro ser trp tyr val Polypeptides and proteins • Here is an example sequence of amino acids in a protein. • It also shows the abbreviations commonly used.

Polypeptides and proteins • Residue - term used to refer to the amino acid once incorporated into a polypeptide • Polypeptide - contain 10-100 residues • Protein - contain more than 100 residues. • Most peptides and proteins isolated from cells contain between 2 - 2000 residues. • An average amino acid has a weight of 110, so protein molecular weights are in the range of 220 - 220,000 (some are much larger).

H O | || H2N - C - C | R H O | || - NH - C - C - | R’ H | N - C - COOH | | H R’’ Peptides N-terminal residue C-terminal residue peptide linkages

Protein function • Enzymes biological catalysts. • Immuno- antibodies of immune system. • globulins • Transport move materials around - • hemoglobin for O2. • Regulatory hormones, control metabolism. • Structural coverings and support - • skin, tendons, hair, nails, bone. • Movement muscles, cilia, flagella.

Protein size, composition and properties • One important property is molecular weight. There are two common ways to calculate it. • Determine the number of amino acid residues, then multiply by 110 -- the average molecular weight of an amino acid. • Directly measure the mass of a protein and report it in daltons. One dalton = One atomic mass unit.

Size of some important proteins • Protein MW Residues • Insulin 6,000 51 • Cytochrome c 16,000 104 • Hemoglobin 65,000 574 • Gamma globulin 176,000 1320 • Myosin 800,000 6100

Protein composition • Proteins can be classified based on the number of polypeptides used • Monomeric - only a single polypeptide chain is present. • Oligomeric - two or more polypeptide chains are present. • The subunit peptide chains are typically held together with noncovalent bonds.

Protein composition • Proteins are also classified based on their composition. • Simple proteins - only contain amino acid residues. • Conjugated proteins - contain other biomolecules - prosthetic groups. • These groups impart additional properties to a protein.

Example - cytochrome C 550 Heme structure Contains Fe2+ Used in metabolism. Aggregate of proteins and other structures.

Protein solubility • Two categories. • Determined by the types of amino acid side chains involved. • Water soluble • - globular proteins • Water insoluble • - fibrous proteins.

Four levels of protein structure • Primary structure • The actual sequence of amino acids in a protein. • Secondary structure • The type of regular repeating structure (-helix, -sheet) • Tertiary structure • Interaction of side chains. • Quaternary structure • Association of two or more polypeptide chains to form a multisubunit molecule.

H O | || H2N - C - C | R H O | || - NH - C - C - | R’ H | N - C - COOH | | H R’’ Summary ofprotein structure primary secondary tertiary quaternary

Determination of primary structure • The first step is to isolate the protein in a pure form from its natural source. Typically, only very small amounts can be obtained. • Total amino acid composition can be determined by hydrolysis of the protein. (6M HCl at 100oC). • The amount of each amino acid can then be measured chromatography.

Protein sequencing • Methods that determine the order of each amino acid in a protein. • Edman degradation. • Method of choice for protein sequencing. • Relies on a sequential degradation by removing one amino acid at a time from the N-terminus. • Process can be automated and works with peptides with up to 50 residues.

- NH COO 2 - COO - NH COO 2 H Edman degradation O O + CH C N CH C N R H R' phenylisothiocyanate peptide H S H O O N C N CH C N CH C N H R R' H+ isolate and react with additional reagent. O CH C N R' phenylthiohyantoin remaining peptide

Edman degradation • Problems with the method. • Does not provide 100% yield - resulting in contamination. • Limited to about 50 cycles so proteins must be cut to smaller sizes. Must rely on enzymes and reagents to cleave a protein at known locations. • Disulfide bonds between cysteine residues can present problems.

Protein sequencing • As of 1998, over 30,000 protein sequences were available in a computer database. • Having such information available makes it possible to study and compare sequence information. • Several biochemical conclusions have been made as a result of studying this data.

Protein sequencing • Identification of protein families. • Proteins with common sequence features have similar biological function, • This allow for the characterization of newly discovered proteins. • Example - protein kinases • Enzymes that catalyze the phosphorylation of amino acid residues. • All known protein kinases have the same common sequence region (domain) of 240 residues.

Protein sequencing • Evolutionary development of proteins • Comparison of protein types for many organisms. • Possible to establish taxonomic relationships. • Example - cytochrome c • Protein used in aerobic respiration. • It has been determined for over 60 organisms. • 27 residues are the same for all forms. • Other variations indicate evolutionary changes.

Protein sequencing • Search for dysfunction. • Normally, all residues in a protein are identical for a species. • Some individuals may produce a protein with one or more ‘incorrect’ residues. • Example - sickle cell anemia. • Two ‘incorrect’ amino acid residues result in malformed hemoglobin. • This causes deformation of red blood cells.

Protein sequencing • Three dimensional nature of proteins. • Sequence data can be coupled with other methods. • X-ray crystallography can produce 3-D structural information. • It is a difficult method and has not kept up with the number of proteins that have been isolated. • Sequencing may offer an alternative approach.

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Protein sequencing • Example - ribonuclease

Chromatography and electrophoresis of proteins • For a protein to be assayed by X-ray crystallography or protein sequencing, a pure sample must be produced. • After preparation of a cell extract, an appropriate separation method must be employed. Two such methods are: • Chromatography • Electrophoresis

Chromatography • Several chromatographic methods have been attempted to isolate pure protein fractions. • ion exchange • thin layer chromatography • column liquid chromatography • size exclusion chromatography • affinity chromatography • Affinity chromatography is becoming increasingly more important.

Affinity Chromatography • The method dates back to 1910. • Modern method was first published in 1967, by Axen, et al. -- ‘Cyanogen bromide Method for the Immobilization of Ligands on Agarose.’ • Ohlson (1978) was the first to demonstrate the use of a rigid, microparticulate support - beginnings of instrumental method.

Affinity Chromatography • The method involves the interaction of a ligand with the solute of interest. It can be viewed as being comparable to ion-exchange. • Two general types of ligands • Specific Binds only to one species. • Antibody/antigen • General Group specific • Binds to specific groups • on target species.

Affinity chromatography • Support • The material that the ligand is bound to. • Ideally, it should be rigid, stable and have a high surface area. • Agarose is the most popular although cellulose, dextran and polyacrylamide have been evaluated.

Affinity chromatography • Agarose gel • A polymer of D-galactose and 3,6-anhydro-L-galactose. • It can be used at pressures up to 1 psi and over a pH range of 4-9. • Cross-linking can be used to extend the pressure range.

Affinity chromatography • The separation is conducted in four basic steps. • Sample introduction • Adsorption of components of interest • Removal of impurities • Elution of components.

Affinity chromatography • Sample introduction • You must make sure • that your column has adequate capacity. ligand matrix spacer

Affinity chromatography • Absorption • Using a slow flow, your sample is then allowed to pass through the column. • The flow helps drive your sample components towards ‘fresh’ sites.

Affinity chromatography • Washing • Next, you can remove impurities by passing several volumes of fresh solvent through the column.

Affinity chromatography • Elution • The component of interest must then be removed and collected. • This also acts to regenerate the column.

Affinity chromatography • Elution methods • Biospecific • An inhibitor is added to the mobile phase (free ligand). • The free ligand will compete for the solute. • This approach is most often used when a low molecular weight inhibitor is available.

Affinity chromatography • Elution methods • Nonspecific • A reagent is added that denatures the solute (pH, KSCN, urea, ionic strength...) • Once denatured, the solute is released from the ligand. • If the solute is to be further used, it must not be irreversibly altered.

Affinity chromatography An example. Column: 50 mm x 30 mm containing 60 ml of Protein A Sepharose Sample: 5 liter cell culture supernatant with mouse IgGa2 and 0.5% fetal calf serum. Starting buffer: 0.1 M Na2HPO4, pH 7 Elution buffer: 0.1 M citric acid, pH 4 Flow rate: 66.6 ml/min 60 70 80 minutes

Electrophoresis • A separation method that relies on both the size and the charge of a species. • Samples are placed in an electrical field. • They tend to migrate to specific positions in the field. • With gel electrophoresis, a cross-linked polymer acts like a molecular sieve - smaller proteins move faster than larger ones.

Amino Acids, Peptides and Proteins