Part 1.

Amino acid A compound that contains both an amino group and a carboxyl group o o o A-Amino acid has an amino group attached to the carbon adjacent to the carboxyl group -carbon also bound to side chain group, R R gives identity to amino acid

Essential Amino Acids 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Isoleucine Leucine Arginine Phenylalanine Methionine Tryptophan Histidine Lysine Threonine Valine

Two stereoisomers of amino acids are designated Lor DThree-Letter Abbreviation Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val One-Letter Abbreviation A R N D C E Q G H I L K M F P S T W Y V

Amino Acid Structure and Properties With the exception of glycine, all proteinderived amino acids have at least one stereocenter (the a-carbon) and are chiral (stereoisomers) The vast majority of a-amino acids have the L-configuration at the a-carbon (Proline is usually D) Side-chain carbons in other amino acids designated with Greek symbols, starting at a carbon (etc) Amino acids can be referred to by threeletter or one-letter codes.

Amino Acid Alanine Arginine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Individual Amino Acids Group A: Nonpolar side chains- Ala, Val, Leu, Ile, Pro. Phe, Trp, Met. o Ala, Val, Leu, Ile, Pro- contain aliphatic hydrocarbon group. Pro has cyclic structure. o Phe - hydrocarbon aromatic ring. o Trp - Indole ring side chain, aromatic. o Met - Sulfur atom in side chain. Group B: Neutral Polar side chains- Ser, Thr, Tyr, Cys, Glu, Asn o Ser, Thr- Side chain is polar hydroxyl group o Tyr- hydroxyl group bonded to aromatic hydrocarbon group o Cys- side chain contains thiol group (-SH) o Gln, Asn- contain amide bonds in side chain Group C: Acidic Side Chains: Glu, Asp o Both have a carboxyl group in side chain o Can lose a proton, forming a carboxylate ion o These amino acids are negatively charged at neutral ph Group D: Basic side chains: His, Lys, Arg o Side chains are positively charged at ph 7 o Arg-side chain is a guanidino group o His-side chain is an imidazole group o Lys-side chain NH3 group is attached to an aliphatic hydrocarbon chain

Classification of Amino Acids based on side-chain reactivity and polarity at pH 7.4: Hydrophobic Polar, Polar, Charged Uncharged Gly Ser Asp Ala Thr Glu Val Cys Lys Leu Tyr Arg Ile Asn Pro Gln Met His Phe Trp

Important structural features: 1. 2. 3. 4. 5. All 20 are a-amino acids For 19 of the 20, the a-amino group is primary; for proline, it is secondary With the exception of glycine, the a-carbon of each is a stereocenter Isoleucine and threonine contain a second stereocenter 3, and 1-letter codes

Henderson-Hasselbalch Equation We have calculated the ratio of acid to conjugate base for an a-carboxyl group and an a-amino group at ph 7.0 We can do this for any weak acid and its conjugate base at any ph using the Henderson-Hasselbalch equation

Isoelectric ph Electrophoresis The process of separating compounds on the basis of their electric charge o Electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose acetate as solid supports o In paper electrophoresis, a paper strip saturated with an aqueous buffer of predetermined ph serves as a bridge between two electrode vessels The ph at which the majority of molecules of a compound in solution have no net charge

Uncommon Amino Acids Each derived from a common amino acid by a modification a. Hydroxylysine and hydroxyproline are found only in a few connective tissues such as collagen b. Thyroxine is found only in the thyroid gland

Ionization of Amino Acids In amino acid, carboxyl group (-) and amino group (+) are charged at neutral ph. In free amino acids -carboxyl, and a-amino groups have titratable protons. Some side chains do as well Remember, amino acids without charged groups on side chain exist in neutral solution as zwitterions with no net charge

Titration of Amino Acids The Peptide Bond When an amino acid is titrated, the titration curve represents the reaction of each functional group with the hydroxide ion Covalent bond that joins amino acids together in a condensation reaction Formed between the alpha-amino group of one amino acid and the alpha-carboxyl group of another amino acid Resonance interaction of the N lone pair with the C=O of the carbonyl in the amide results in the C-N bond having some double bond character (shorter, restricted rotation) Planar sp2 (120 bond angles) N system allows the N lone pair to align with the C=O p system Peptide bond formation amide synthesis Structure : o Double bond character of the C-N unit makes the structure less flexible o Planarity and the anti-conformation are important in dictating protein shape Reactivity : o Resonance donation of electrons by the N makes the carbonyl less electrophilic o The amide is comparatively unreactive this is a good thing otherwise proteins would be too reactive to be much use in biological systems

Acidity: a-COOH Groups The average pka of an a-carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pka 4.76) The greater acidity of the amino acid carboxyl group is due to the electron-withdrawing inductive effect of the -NH3+ group A-NH3+ groups: The average value of pka for an aNH3+ group is 9.47, compared with a value of 10.76 for a 2 alkyl ammonium ion Guanidine Group o The side chain of arginine is a considerably stronger base than an aliphatic amine Basicity of the guanido group is attributed to the large resonance stabilization of the protonated form relative to the neutral form Imidazole Group o The side chain imidazole group of histidine is a heterocyclic aromatic amine

Basicity

Peptides Peptide: the name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain Dipeptide: a molecule containing two amino acids joined by a peptide bond Tripeptide: a molecule containing three amino acids joined by peptide bonds Polypeptide: a macromolecule containing many amino acids joined by peptide bonds Protein: a biological macromolecule of molecular weight 5000 g/mol or greater, consisting of one or more polypeptide chains

3. Concentration of ions or molecules Urea (nh2conh2) is one of several small molecules which, at high concentrations, can weaken the noncovalent forces keeping the secondary and tertiary structure intact Protein becomes denatured as its structure unravels and produces separate random coils in solution Protein in this state has none of its original biological properties If the urea is removed, denaturation may be reversed as the protein can slowly coil and fold back into its original conformation, regaining all its biological properties Proteins with many disulfide bridges, breaks apart by the use of reducing agents

Protein classification Based on composition o Simple proteins no other biomolecules present o Conjugated proteins presence of metal atom or small organic molecule Based on solubility o Globular water soluble; transport function, immune protection and catalysis o Fibrous water insoluble; structural functions collagen, elastin

4. Temperature Increasing the temperature causes atoms to move about more quickly change in the shape of the protein

Levels of Protein Structure 1structure: the sequence of amino acids in a polypeptide chain, read from the N-terminal end to the C-terminal end 2structure: the ordered 3-dimensional arrangements (conformations) in localized regions of a polypeptide chain; refers only to interactions of the peptide backbone o e. g., a-helix and b-pleated sheet 3 structure: 3-D arrangement of all atoms 4 structure: arrangement of monomer subunits with respect to each other

Part 2. Protein Structure Many conformations are possible for proteins: o Due to flexibility of amino acids linked by peptide bonds At least one major conformations has biological activity, and hence is considered the proteins native conformation

Factors affecting protein conformation 1. pH Small changes in pH can add or remove h+ ions from side chain groups on the surface of a protein

Protein Primary Structure (1 Structure) sequence of amino acids that make up a protein determines 3-dimensional structure

Importance of primary structure Determines the 3-dimensional arrangement of proteins/folding/stability Determines the solubility of the protein Determines the electrophoretic mobility of proteins Determine evolutionary relationships among species

2. Polarity of the solvent At a certain pH, the isoelectric point, the protein molecule will have no overall ionic charge and so will have its minimum solubility in water Different proteins have different isoelectric points use this to precipitate one protein from a mixture of proteins in solution by adjusting the pH to the isoelectric point of that protein

Protein Secondary Structure (2 Structure) Arrangement of the peptide backbone in space (conformation) Refers to regular, repeated patters of folding of the protein backbone Patterns result from regular hydrogen bond patterns of backbone atoms phi = 0, psi = 180 is unfavorable phi = 180, psi = 0 is unfavorable phi = 0, psi = 0 is unfavorable Two most common folding patterns are the alpha helix and the beta sheet 1. -helix o Peptide is coiled around an imaginary cylinder (like a telephone cord) o Stabilized by hydrogen bonds formed between components of the peptide bonds o Hydrogen bonds between the c=o of residues n and the nh of residues n+4 o 3.6 amino acid residues per helical turn; rises 1.5 a per residue o Right-handed helix, amino acid r- groups orient out o Average helix is 10 residues long (15 a in length), although can range between 4 to 40 residues in length o Some amino acids are preferred in an helix: residues such as ala, glu, leu and met have a high tendency to participate in a helix; of special interest is proline, which cannot fit into a helix, and introduces a kink 2. -pleated sheet o o o o formed by hydrogen bonding between several -strands not fully extended due to side chain steric interferences extended conformation in a beta-strand is about 3.5 A per residue, i.e., beta-strands can be extended as much as 35 A in length overall geometry of a sheet is not planar but rather it is a pleated sheet alternating C-alpha carbons above and below the average plane of the sheet

o o

a beta-sheet has an overall left-handed twist strands do not have to be adjacent on the sequence, there are many possible ways to arrange strands in a sheet these arrangements are called topologies and can be quite complicated

-Turn

Short secondary structure, only 4 residues in length, enables the overall structure to have 180 degree turns Characterized by a hydrogen bond between the c=o group of residue n and the nh group of residue n+3 (i.e, between the first and the fourth residue of the turn) Has several unsaturated back-bone hydrogen bond donors and acceptors, it is polar, and is usually found near the surface of the protein Gly and pro residues are common in -turns

Random coils Parts of the protein that are not characterized by any regular hydrogen bonding patterns 2 places where random coils can be found: o Terminal arms - both at the n-terminus and the c-terminus of the protein o Loops Unstructured regions found between regular secondary structure elements 4 to 20 residues long, although most loops are not longer than 12 residues Exposed to the solvent and are characterized by polar or charged sidechains In some cases loops have a functional role, but in many cases they do not

a-Helices and -Sheets Supersecondary structures: the combination of aand b-sections, as for example o o o a unit: two parallel strands of b-sheet connected by a stretch of a-helix aa unit: two antiparallel a-helices -meander: an antiparallel sheet formed by a series of tight reverse turns connecting stretches of a polypeptide chain Greek key: a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itself -barrel: created when -sheets are extensive enough to fold back on themselves

3 possible ways to form a beta-sheet from beta-strands: Parallel beta-sheet - have the same N to C direction (both chains running N-terminal to C-terminal in the same direction); hydrogen bonds are equally distanced. Antiparallel beta-sheet have opposite N to C direction Mixed beta-sheet - a mixture of parallel and antiparallel hydrogen bonding; mixed sheets are about 20% of all beta-sheets o due to the chirality of the amino acids, all betastrands have a right-handed twist o

Collagen Triple Helix o Consists of three polypeptide chains wrapped around each other in a ropelike twist to form a

o o o o

triple helix called tropocollagen; MW approx. 300,000 30% of amino acids in each chain are Pro and Hyp (hydroxyproline); hydroxylysine also occurs Every third position is Gly and repeating sequences are X-Pro-Gly and X-Hyp-Gly Each polypeptide chain is a helix but not an ahelix The three strands are held together by hydrogen bonding involving hydroxyproline and hydroxylysine With age, collagen helices become cross linked by covalent bonds formed between Lys and His residues

o o o o

Non-regular secondary structures Bends/loops they reverse direction of the main polypeptide; they connect regions of more regular secondary structures o B bends or hairpin turns are common with 4 aa in the structure and one internal H-bond between the 1st and 4th aa; Gly and Pro are prominent in bends Loops - extended bends between 6 to 16 amino acids

Arrangement of polypeptide subunits within complex proteins made up of two or more subunits, sometimes associated with non-protein groups Non-covalent interactions that bind multiple polypeptides into a single, larger protein Hetero- or homo- polymers/monomer, dimers, trimmers, tetramers Same forces drive tertiary and quaternary structure When polypeptide sub-units join together in very large numbers they form supra molecular assemblies Non-covalent interactions o electrostatics, hydrogen bonds, hydrophobic Oligomeric two or more polypeptide chains; subunits Defines the arrangement and position of each subunit in an intact protein Homotypic almost identical subunits Heterotypic different subunits

Protein Folding Chaperones In the protein-dense environment of a cell, proteins may begin to fold incorrectly or may associate with other proteins before folding is completed Special proteins called chaperones aid in the correct and timely folding of many proteins hsp70 were the first chaperone proteins discovered Chaperones exist in organisms from prokaryotes to humans

Protein Tertiary Structure (3 Structure) tertiary structure of a protein is the arrangement in space of all its atoms the final three-dimensional structure of a protein, which results from a large number of non-covalent interactions between amino acids overall 3-dimensional shape of a protein molecule is a compromise, where the structure has the best balance of attractive and repulsive forces between different regions of the molecule determined and stabilized by chemical bonds and forces, including weak bonds (Hydrogen bonds, Ionic bonds, Van der Waals bonds, and Hydrophobic attractions) Amino acids which are very distant in the primary structure might be close in the tertiary structure because of the folding of the chain. Domains The part of a polypeptide chain that independently fold into a tertiary structure Often have units of function Proteins may contain one or many domains can

Denaturation the loss of the structural order (2, 3, 4, or a combination of these) that gives a protein its biological activity; that is, the loss of biological activity Denaturation can be brought about by: o o o o o heat large changes in pH, which alter charges on side chains, e.g., -COO- to -COOH or -NH3+ to -NH2 detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions urea or guanidine, which disrupt hydrogen bonding mercaptoethanol, which reduces disulfide bonds

Denaturation and Refolding in Ribonuclease o Several ways to denature proteins: Heat pH Detergents Urea Guanadine hydrochloride

Protein Quaternary Structure (4 Structure)

Part 3. Peptide hormones Hormones chemical messengers secreted into blood or extracellular fluid by one cell that affect the functioning of other cells Insulin and glucagon Insulin hypoglycemic hormone; released by the - cells of pancreatic islets in response to hyperglycemia (elevated glucose levels); Glucagon hyperglycemic hormone; released by the cells in response to hypoglycemia (low glucose levels) Transport proteins

Since residues of a beta sheet extend alternately above and below the plane of the sheet, this places all glycines on one side and all alanines and serines on other side! This allows Glys on one sheet to mesh with Glys on an adjacent sheet (same for Ala/Sers)

Myoglobin tertiary structure Hemoglobin quaternary structure

Protein hormones Hormones chemical messengers secreted into blood or extracellular fluid by one cell that affect the functioning of other cells A given hormone usually affects only a limited number of cells target cell responds to a hormone because it bears receptors for the hormone Change in hormone structure may render it unrecognizable by its receptor abnormal biological effects Peptide hormones are synthesized in endoplasmic reticulum, transferred to the golgi and packaged into secretory vesicles for export Peptide and protein hormones vary considerably in size, ranging from peptides as short as 3 amino acids to large, multisubunit glycoproteins Many protein hormones are synthesized as prohormones, then proteolytically clipped to generate their mature form In other cases, the hormone is originally embedded within the sequence of a larger precursor, then released by multiple proteolytic cleavages

Structural Proteins Collagen o Most abundant structural protein o Genetic disorder Osteogenesis imperfecta infants born with fragile bones; Cys/Ser occupies position reserved for Gly o Scurvy vitamin C deficiency characterized by bleeding gums and skin rashes; Vit C is a cofactor for proline hydroxylase which converts proline to hydroxyproline Elastin o Found in connective tissues, large arteries such as aorta, trachea, bronchi and ligaments o Elastic and allows many tissues in the body to resume their shape after stretching or contracting. o Helps skin to return to its original position when it is poked or pinched. o Amino acid composition: glycine, valine, alanine and proline; but contains little hydroxyproline and no hydroxylysine. o Made by linking many soluble tropoelastin protein molecules to make a massive insoluble, durable cross-linked array desmosine crosslinks Keratins o Alpha Keratin Sequence consists of 311-314 residue alpha helical rod segments capped with non-helical N- and Ctermini Primary structure of helical rods consists of 7-residue repeats: (a-bc-d-e-f-g)n, where a and d are nonpolar. Promotes association of helices. o Beta Keratin 1o structure: alternating sequence: Gly-Ala/Ser-Gly-Ala/Ser

Insulin Produced by cells of the pancreatic islets Composed of 2 peptide chains: a chain and b chain linked together by 2 disulfide bonds (interchain) Additional disulfide (intrachain) is formed within the a-chain In most species, the a chain consists of 21 amino acids and the b chain of 30 amino acids Amino acid sequence of insulin varies among species, certain segments of the molecule are highly conserved positions of the 3 disulfide bonds, both ends of the a chain and the c-terminal residues of the b chain Similarities in the amino acid sequence of insulin lead to a 3-dimensional conformation of insulin that is very similar among species, and insulin from one animal is very likely biologically active in other species

Opposing effects of insulin and glucagon

Insulin hypoglycemic hormone released in response to hyperglycemia (elevated glucose levels) Glucagons hyperglycemic hormone released in response to hypoglycemia (low glucose levels)

Globular proteins Far outnumber fibrous proteins Perform most of the chemical "work" of the cell synthesis, transport, and catabolism Folded into compact structures very unlike the extended, filamentous forms of the fibrous proteins 3o structure of globular proteins reflects their interaction with their aqueous solvent = o Consist of a hydrophobic core surrounded by a hydrophilic external surface which interacts with water o Fold of the polypeptide is such that those residues with apolar side chains are buried. In the center, while the polar residues remain exposed o Dominant driving force behind the folding of the polypeptide chain into the compact Globular form: the aggregation and burial of the hydrophobic surface reduces the Number of unfavorable interactions of these groups with water: the hydrophobic Effect

nitrogen of histidine f8 (or proximal histidine: residue number 93, located in f helix) In deoxymyoglobin, the remaining coordination site, on the other side of the fe occupied by a water molecule When oxygen is bound, making oxymyoglobin, O2 displaces the water molecule On the other side of the bound O2 lies histidine e7 (or distal histidine: residue 64, located in e helix)

Oxygen binding by myoglobin Non-cooperative binding of O2 O2 binding curve of myoglobin in solution at neutral pH: Illustrates how the fraction of the myoglobin sites that have oxygen bound to them ( ) Depends on the concentration (partial pressure) of free oxygen Hyperbolic: p50 (oxygen partial pressure required for half saturation) is very low = myoglobin has a high affinity for oxygen = important characteristic for a protein that must extract oxygen from the small amounts present in blood Hemoglobin Tetrameric (2, 2 chains) heme-protein In erythrocytes, responsible for binding o 2 in the lungs and transporting the bound o2 throughout the body where it is used in aerobic metabolic pathways Each subunit has a heme prosthetic group identical to that described for myoglobin Binds a total of 4 O2 molecules to its 4 heme groups The closest and strongest contacts appear to be between and chains, rather than - or 2o and 3o structure of subunits are similar extensive homology in amino acid composition Variations in amino acid composition do exist = marked differences in Hemoglobin's o2 carrying properties 4o structure leads to physiologically important allosteric interactions between the subunits, a property lacking in monomeric myoglobin Hemoglobin in blood is bound to BPG Interaction is electrostatic, between negative charges on BPG and positive side chains (e.g., Lys, Arg) of hemoglobin BPG promotes O2 dissociation Hb stripped of BPG remains saturated with O2 Types of hemoglobin: o Carboxyhemoglobin Hb + CO o Carbaminohemoglobin Hb + CO2 o Oxyhemoglobin Hb + O2 o Deoxyhemoglobin Hb - O2 o Ferrohemoglobin Hb + Fe2+ o Methemoglobin Hb + Fe3+

Myoglobin Monomeric heme protein Oxygen storage protein (binds 1 oxygen molecule per molecule of protein) Oxygen transported to tissues must be released for utilization in tissues, such as muscle, with high oxygen demands, myoglobin provides large oxygen reserves 3o structure is that of a typical water soluble globular protein Unusual in that it contains a very high proportion (75%) of -helical secondary structure Myoglobin polypeptide comprised of 8 separate right handed -helices, designated a through h, connected by short non helical regions The single polypeptide chain folded about a prosthetic group, the heme, which contains the oxygen binding site o Heme is in the form of an iron-complex with protoporphyrin Heme noncovalently bonded in a hydrophobic crevice in the myoglobin or hemoglobin molecule Ferrous iron is normally octahedrally coordinated 6 ligands, or binding groups, attached to it: nitrogen atoms of the porphyrin ring account for 4 of these ligands; 2 remaining coordination sites are available, lie along an axis perpendicular to the plane of the ring In both the deoxygenated and the oxygenated forms of myoglobin, one of these sites is occupied by the

Oxygen binding by hemoglobin

The effect of pH on the oxygen-binding ability of Hb is called the Bohr effect O2 binding curve of hemoglobin in solution at neutral pH: Sigmoidal: typical of allosteric proteins in which the substrate, in this case oxygen, is a positive homotropic effector

First line of defense against infection (bacterium, virus, foreign protein) and probably against cancer cells as well Foreign substance that elicits an immune response = antigen Specific immunoglobulin that binds to the antigen = antibody Immune response has a so-called memory

Allosteric effects and cooperatives Allosteric effects occur when the binding properties of a macromolecule change as a consequence of a second ligand binding to the macromolecule and altering its affinity towards the first, or primary, ligand 2 ligands are the same (e.g. Oxygen) = homotropic allosteric effect 2 ligands are different (e.g. Oxygen and bpg) = heterotropic allosteric effect Macromolecules that have multiple ligand binding sites (e.g. Hb), allosteric effects can generate cooperative behavior Part 4. Isolation of proteins Based on molecular size Based on charge Based on solubility Many steps needed to extract protein of interest, and separate from many contaminants Before purification begins, protein must be released from cell by homogenization

Chromatography Separation technique based on the difference between the affinities of substances in a mobile phase and a stationary phase Components: mobile phase (liquid or gas); stationary phase (liquid or solid) and a inert solid support

Clinical conditions resulting from abnormal hemoglobin: Sickle cell hemoglobin Thalassemia

Immunoglobulin foreign substance that elicits an immune response = antigen specific immunoglobulin that binds to the antigen = antibody invading particle is large, like a cell, a virus, or a protein, many different antibodies may be elicited, each type binding specifically to an antigenic determinant (or epitope) on the surface of the particle 5 classes of immunoglobulin molecules (IgM, IgA, IgG, IgD & IgE) which carry out various functions in the immune system different kinds of antibodies may contain from 1 to 5 immunoglobulin molecules more than one is present, the monomers are linked by a second type of polypeptide, called a J chain Y-shaped 2 identical binding sites for its antigen, one on either arm of the Y composed of 4 polypeptide chains (2 identical heavy chains and 2 identical and smaller light chains) held together by disulfide bonds each chain is made up of several different domains antigen-binding site is formed where a heavy chain variable domain (VH) and a light chain variable domain (VL) come close together domains that differ most in their sequence and structure in different antibodies The immune response

Column Chromatography Basis of Chromatography Different compounds distribute themselves to a varying extent between different phases Interact/distribute themselves In different phases 2 phases: o Stationary: samples interacts with this phase o Mobile: Flows over the stationary phase and carries along with it the sample to be separated

Chromatographic methods in protein isolation Size exclusion chromatography o based on the ability of molecules to move through a column of gel that has pores of clearly-defined sizes. The larger molecules can not enter the pores, thus they pass quickly through the column and elute first. smaller molecules can enter some pores, and so take longer to elute, and small molecules can be delayed further.

o o

Stationary phase composed of cross-linked gel particles. Extent of cross-linking can be controlled to determine pore size

Charged particles migrate in electric field toward opposite charge Proteins have different mobility: o o Charge Size Shape

Ion exchange chromatography o o Based on the ability of the molecules to pass through a column of charged resin Resins with positively charge groups anion exchangers; bind negatively charged molecules Resins with negatively charge groups cation exchangers; bind positively charged molecules

Agarose used as matrix for nucleic acids Polyacrylamide used mostly for proteins Polyacrylamide has more resistance towards larger molecules than smaller Protein is treated with detergent (SDS) sodium dodecyl sulfate Smaller proteins move through faster (charge and shape usually similar)

Gel electrophoresis Affinity chromatography o o Uses specific binding properties of molecules/proteins Based on the ability of the molecules to pass through a column of matrix with chemical groups or ligands to which the molecule can specifically bind Based on charge and molecular size Support medium is cellulose or thin gels made up of either polyacrylamide or agarose. Cellulose is used as support medium for low molecular weight biochemicals such as amino acid and carbohydrates whereas agarose for large molecules like proteins SDS PAGE o proteins are denatured to make them have a uniform charge, electrophoretic mobility depends primarily on size

Membrane separation methods Dialysis o based on molecular size

Membrane filtration/Ultrafiltration o Is a low-pressure membrane process used to separate high molecular weight compounds from a feed stream. Ultrafiltration has larger pores than nanofiltration and reverse osmosis and is therefore the least costly of the three to operate. Ultrafiltration is useful for the separation of delicate materials since it is a non-denaturing method of separation.

Isoelectric Focusing based on differing isoelectric pts. (pI) of proteins Gel is prepared with pH gradient that parallels electric-field. Effects: o Charge on the protein changes as it migrates. o When it gets to pI, has no charge and stops

Precipitation methods Isoelectric precipitation o pH of solution is adjusted to the IpH of the protein; at this pH the protein is least soluble

Differential centrifugation Sample is spun, after lysis, to separate unbroken cells, nuclei, other organelles and particles not soluble in buffer used Different speeds of spin allow for particle separation

Salting out methods o o addition of different concentration of salt like (NH4)2SO4; proteins are desolvated After Proteins solubilized, they can be purified based on solubility (usually dependent on overall charge, ionic strength, polarity Takes away water by interacting with it, makes protein less soluble because

Ultracentrifugation A high-velocity centrifuge used in the separation of colloidal or submicroscopic particles. With appropriate photography of the protein layers as they form in the centrifugal field, it is possible to determine the molecular weights of proteins.

Electrophoresis

hydrophobic interactions among proteins increases o Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest (30, 40, 50, 75% increments) One fraction has protein of interest

Step 3 Determine amino acid composition Chemical (acid) nad enzymatic (peptidases) hydrolysis of polypeptides followed by analysis of amino acid Amino acid composition analyzed by HPLC

Protein Cleavage Protein cleaved at specific sites by: o Enzymes - Trypsin, Chymotrypsin o Chemical reagents - Cyanogen bromide Enzymes: o Trypsin - Cleaves at C-terminal of (+) charged side chains o Chymotrypsin - Cleaves at C-terminal of aromatics CNBr o Cleaves at C-terminal of internal methionines

Step 4 Step 5 Cleave each chain into smaller fragments and determine the sequence of each chain Enzymatic fragmentation o Trypsin Cleavage on the C-side of Lys and Arg o Chymotrypsin Cleavage on the C-side of Phe, Tyr and Trp o Elastase Cleavage on the C-side of neutral residues o Chemical fragmentation Cyanogen bromide Cleavage on the C-side of Met End group determination N-terminus determination o Dansyl chloride reacts with primary amines C-terminues determination o Carboxypeptidase or other exopeptidases

Amino acid sequencing Hydrolysis HPLC Sanger analysis N terminal Amino acid Edman degradation Use of specific enzymes and chemical reagents Look for overlapping sequences Step 6 Step 7 Step 1 If more than one polypeptide chain, separate Subunit interactions depend on weak forces Separation is achieved with: o Extreme pH o 8M urea o 6M guanidine HCl o High salt concentration [(NH4)2SO4] Step 8

Determining Protein Sequence After cleavage, mixture of peptide fragments produced. Can be separated by HPLC or other chromatographic techniques Use different cleavage reagents to help in 1 determination

Repeat step 5, using a different cleavage procedure to generate a different set of fragments Reaction with Trypsin o Separate peptide fragments Reaction with Chymotrypsin o Separate peptide fragments Reaction with CNBr o Separate peptide fragments

Protein sequencing Sequence all the peptides produced usually by Edmand degradation

Reconstruct the sequence of the protein from the sequences of overlapping fragments Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain

Step 2 Cleavage of disulfide bridges by chemical reagents