Proteins

Protein classification

• any combination of 20 amino acids joined together by amide bonds

• the sequence influences the structure and function

• mad ein ribosomes and may or may not be modified further

two classifications of proteins

• simple proteins (homoproteins)

• Conjugated proteins (heteroproteins)

Simple proteins (homoproteins)

• polypeptide chains containing only amino acids and no further changes after synthesis

• classified further by solubility in water

Simple sugars dissolvable

• albumins dissolve in pure water

• egg albumin

• lactalbumin in milk

Simple sugrs non soluble

• Globulins dissolve in a dilute salt solution

• gluten

• myosin and actin in muscle

Conjugated proteins (heteroproteins)

• proteins that have been modified by cytoplasmic enzymes or attached with non-protein functional groups

• functional groups include carbohydrates, phosphate, nucleic acid, lipids, metal ions

• includes

  • Glycoproteins

  • phosphoproteins

Glycoproteins

• conjugated proteins covalently linked to carbohydrate groups

• oligosaccharide chains are covalently linked to the amino acid side chains of the protein

  • ovotransferrin and ovomucoid (egg white proteins)

Phosphoproteins

• congjugated proteins that are covalently linked to phosphate groups

• phosvitin → egg yolk protein

Structural proteins

• make up the structural parts of a body like bone, muscle, skin and cartilage

Collagen

• most common animal protein found in connective tissues

• long cylindrical protein made up of 3 twisted polypeptide chains in a helix

• Hydrogen bonds join them end to end to form long fibres

Gelatin

• made of heated collagen treated with an acid or base to partially hydrolyze the collagen

• the helical structure of collagen becomes more unstructured and amorphous

Gelatin and temperature

• if temperatures drop below 35 degrees, the sections of collagen will partially renature to form helical structure and begin to fold onto itself to H-bond with other renatured sections

  • this forms junction zones to make a 3D gel structure

• Temperatures above 35 degrees will cause the gel to fall apart and collagen will return back to a linear form which cannot H bond

Uses of collagen

• used in jello

• marshmallows to keep soft

• gummy candies for chewy.

• candy coating so that they dissolve slowly

• canned ham

• luncheon meats

• made from animals so the use is restricted

Protein solubility is

• dependent on pH and ionic strength of a solvent

• also dependent on temperature

Soluble protein uses

• thickening agents

• foaming agents

• emulsifying and gelling

pH dependence

• protein charged groups can bind water

  • pH effects the ability of proteins to bind water

• water holding capacity is dependant on aa makeup

  • increase in charged residues increases water binding capacity

water binding capacity

• dependent on pH make up

• the number of charged residues increases the water binding capacity

pH above the isoelectric point

• the protein will be negatively charged

  • and they will repel eachother

• this will increase the water binding capacity

pH below the isoelectric point

• protein is positively charged and the proteins will again repel eachother

  • this increases the water binding capacity

pH equal to isoelectric point

• the protein solubility will be the lowest because there are equal positive and negative charges of the protein and therefore no electrostatic repulsion

• proteins bind eachother, aggregate and precipitate

Solubility curve with pH

• forms a U-shaped curve because of the pH dependance

• the water solubility will be high at low or high pH but not at the pH equivalent to the iso electric point

ionic strength

• salting in= <0.5M salt concentrations

  • charged groups on proteins bind anions and cations more strongly than they bind water

  • ions bring solvating water molecules and keep their protein in solution to increase solubility

• 0.5-1M salt concentrations, the salt ions also prevent the charged groups of the proteins from attracting each other, also increasing solubility

• Salting out = >1M salt concentration

  • decreased protein solubility

  • too many salt ions competing with the protein for the water necessary for the protein to remain in solution

  • salt binds the water and the proteins aggregate and fall out of solution

Salting in

• <0.5M salt concentrations

• charged groups on the protein bind anions and cations more strongly than the bind water

• the ions bring solvating water molecules therefore keeping the protein in solution and increased protein solubility

  • eg. sodium chloride is added to pork making ham

    • adds weight, flavour etc

Middle salt concentrations

• 0.5-1M

• salt ions prevent the charged groups from attracting eachother so solubility is increased

salting out

• too much salt has been added >1M

• there is decreased protein solubility

• too many salt ions have been added which compete with the protein for the water necessary for the protein to remain in the solution

• salt binds the water and the proteins will aggregate and fall out of the solution

Bioactive peptides

• peptides that form part of the protein an which usually have no biological activity

• can be released by heat, acid or base, or microbial and/or enzymatic proteolysis

• positively or negatively influence biological systems

Protein digestion (traditional gastrointestinal digestion pattern)

• after swallowing foods, digestive enzymes break protein into smaller components

Stomach digestion

• Pepsin hydrolysis

• cleavage of the n-terminal end of aromatic AA causing large oligopeptides

Small intestine digestion

• Trypsin and chymotrypsin

  • cleave oligopeptides into di or tri peptides and free AA

• Peptidases

  • digest di/tri peptides into free AA

Proteins not broken down

• some AA and oligopeptides are transported intact across the intestinal epithelial cell to reach the bloodstream

Peptide transport routes 3 carriers

• PepT1 Carrier mediated

• Paracellular

• Transcytosis

PepT1 carrier mediated transport

• proton dependent transporter

• moves peptides from the gut lumen across the intestinal epithelial cells to the basolateral side and into the bloodstream

• only di and tri peptides

  • some fully intact and others are cleaved

Paracellular

• transports between the tight junctions

• tight junctions are the controllers of the paracellular transport

  • composed of claudin proteins which connect adjacent epithelial cells and control the peptide diffusion between cells

• claudin interact to form pores that restrict peptide size

• Transports oligopeptides

  • all are intact

transcytosis

• peptides are transported from the luminal side to the basolateral side

• endocytosis on the luminal side

• vesicle is transported to the other side and fuses witht eh cell membrane

• there is exocytosis of the peptide follows

• Basic and hydrophobic peptides

  • very few are intact and over 90% are hydrolyzed into amino acids

New bioactive peptides

• produced from egg, milk, cereal and fish proteins

• using heat, acid or base hydrolysis or microbial enzymes

• microbial enzymes have different AA cleavage sites than digestive enzymes

• the bioactive compounds may be able to survivve digestion and transport

• once they enter the bloodstream and travel to the target site they can directly affect cells by interacting with molecules that interfere with gene expression

Mechanisms of action of bioactive peptides

• direct interaction

  • antihypertensive peptides

  • antimicrobial peptides

  • mineral binding peptides

• Interference with gene expression

  • epigenetic modification

  • indirect influence on transcription factor activity

  • direct binding of peptide ligand to cell signalling receptor

Antihypertensive peptides - direct molecule interaction

• binding and decreasing the activity of angiotensin-converting enzyme (ACE)

ACE

• angiotensin converting enzyme

• involved in regulation of blood pressure

• peptides from wheat, peas, mushrooms, walnuts, dates

  • MOST STEADY IS SOYBEANS

• peptides inhibit ace in a competitive and non competitive manner

  • both lower blood pressure by inhibiting ace

Competitive ACE inhibiting

• peptides with 2-12 AA competitively interact with the enzymes active site to prevent substrate binding

non-competitive ACE inhibiting

• peptides bind with sites other than the active site which affects the binding of the substrate

Antimicrobial peptides

• peptides from milk lactoferrin with antimicrobial activity agains pathogenic and spoilage microorganisms

• peptides attach to the bacterial cell surface and disrupt th emembrane function resulting in cell death

Mineral binding proteins

• bind and solubize mineral ions so that it is readily available for intestinal uptake

• Casein phosphopeptides (CPPs)

  • 3 continuous phosphoserines then 2 glutamic acid AA

  • solubize calcium and facilitate absorption in the intestine

• Phosvitin phosphopeptides (PPPs)

  • hen egg yolk

  • promote calcium and iron absorption in the intestinal tract

• increased uptake of minerals like calcium is important for osteoblast cells

• promote bone deposition to prevent osteoperosis

Protein interference with gene expression

• epigenetic modification

• indirect influence on transcription factor activity

• direct binding of peptide liand to cell signalling receptor

Casien phosphopeptides

• 3 continuous phosphoserines then 2 glutamic acid AA

• solublize calcium and facilitate absorption in the intestine

phosvitin phosphopeptides (ppps)

• from hen egg yolk

• promotes calcium and iron absorption in the intestinal tract

• increases uptake of minerals like calcium is important in osteoblast cells

• promotes bone deposition and to prevent osteoperosis

Gene expression

• dna is wrapped around histones to form chromatin

  • condenses to chromosomes

• DNA promotor regions are the on switch

• histone modification including acetylation causes the histones to unravel

• transcription factors (TF) bind to the DNA promoter and RNA polymerase or other TF to regulate how much mRNA is produced

Epigenetic modification

• regulation of growth, differentiation and apoptosis

• histone acetylation

  • histone acetyl transferase (HAT) enables the attachment of an acetyl functinoal group to the histone protein in the chromatin

• once the acetyl group binds it leads to transcroption activiation

epigenetic modification example

• Lunasin: soyben polypeptide with structure similar to chromatin binding proteins

  • stops cell dividion by inducing cell death in new cancer cells

• attaches to deacetylated histones in newly changed cells or inhibits histone acetylation catalyzed by HAT proteins

• distruption of histone acetylation- deacetylation is not normal → cell death

• normal non-cancer cells or cells already affected by cancer are not targeted

Lunasin

• soybean protein with part of structure similar to chromatin binding proteins

• stops cell division and induces death in newly transformed cancer cells

• attaches to deacetylated histones or inhibits histone acetylation by HAT proteins

  • this reduces the histone acetylation is abnormal and the cell is killed

  • this way the odd cells cannot continue to proliferate

Process of lunasin epigenetic modification

• E2F/DP is a protein complex which binds to the DNA promotor region (GO)

  • Rb is a tumor supressor which binds to this and recruits histone deacetylase to ensure that histones cannot be acetylated

• RB/HDAC complex keeps the histones regular so Lunasin does not bind

• E1A is a viral protein that causes cancer

  • E1A disrupts the interactions between Rb and HDAC so that HDAC cannot bind (preventing the acetylation preventor therefore allowing deacetylation)

• If LUNASIN binds, they compete with HAT to deacetylate the histone

• If HAT binds, the histones get acetylated and cell division occurs

  • Cancer cells have HAT bound and histones are acetylated so lunasin cannot bind whcih is why they only prevent cancer and cannot get rid of caners

Indirect influence with transcription factor activity

• molecules bind to receptors on the surface or within the cell to trigger a cell signalling response which activates transcription factors

• soy bioactive peptides influences the amount of insulin and glucagon hormones binding to the receptor so some genes are activated compared to others

Sterol regulators element binding proteins (indirect TF influence)

• precursors in the endoplasmic reticulum

• low sterols or insulin in the cytoplasm: the SREBP cleavage activating protein (SCAP) takes the SREBP precursor to the golgi where the SREBP becomes an active protein that can move into the nucleus and bind sterol response elements in the promotor region of genes involved in cholesterol, fatty acid and triglyceride production

  • Insulin promotes SREBP-1 and glucagon suppresses- activates fat synthesis genes

• Low blood cholesterol promotes SREBP-2 whcih activates cholesterol synthesis genes

• soy peptides represses expresion of SREBP-1 so that there is reduced expression of genes in fat synthesis

  • cause an increase in SREBP-2 so there is increased cholesterol uptake and synthesis

Direct binding of peptide ligand to cell signaling receptor

• triggers a cell signalling event that activate transcription and gene expression

• Casein phosphopeptides (CPP) stimulates immune cells and immunoglobulins like IgA to enhance immunity in the gut

Direct binding of peptide to cell signal receptor CPP example

• when bacterial lipopolysaccharide (LPS), CPP interacts with toll-like receptor 4 (TLR-4) on B cells to activate the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-KB) pathway

  • this increases the expression of interleukin-5 and 6 which interact with B cells to promote proliferation and differentiation into plasma cells which release IgA or IgG

  • IgA is important fo rintestinal immune defence to enhance gut mucosal immunity

Sweet tasting amino acids and peptides

• Aspartame

• advantame

• Thaumatin

• Monellin

Aspartame

• L-aspartyl-L-phenylalanine methyl ester

• can be hydrolyzed into L-phenylalanine, L-aspartic acid, and methanol upon digestion

• Contains 4 calories/g

• 200x relative sweetness compared to sucrose

• individual AA which make up aspartame are not sweet

  • when combined htey can create a sweet taste

  • it is a clean taste close to sucrose

  • synergistic with other sweeteners

Stability of apsartame

• hydrolyzed >70 degrees and sweetness gets lost

• unstable outside pH range of 3-6

Issue with aspartame sweetener

• phenylketonuria individuals cannot metabolize L-phenylalanine so indigestion can be hazardous

Advantame

• analogue to aspartame

  • has a chemical substitution on one of the nitrogens

• contains phenylalanine and aspartic acid

• derived from aspartame and vanillin

• 20,000x sweeter than sucrose

• heat stabel and approved in canada

  • in baked goods, yogurts diet sodas