Proteins - Lecture Notes (Topic) PDF

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Proteins • proteins are composed of amino acids linked together in long chains by peptide bonds ! • Amino acids are made up of carbon (50-55%), hydrogen (6-7%), oxygen (20-30%) and nitrogen (12-19%)! • Some amino acids also contain sulphur ! • Most animal proteins contain average about 16% nitrogen ! • A nitrogen to protein conversion factor, 100/16 = 6.25! • Milk proteins 15.67% nitrogen (100/15.67 = 6.38)! • Wheat flour 17.54% nitrogen (100/17.54 = 5.7)! • Gelatine 18.02% nitrogen (100/18.02 = 5.55)! • Proteins play an important role as nutritional, chemical and structural components in food ! • Essential amino acids which cannot be synthesises must be supplied through food proteins for numerous metabolic functions and for growth! • For humans, 9 of the 20 amino acids are essential! • Histidine, isoleucine, leucine, lysine, methionine, phenylalanine, theronine, tryptophan and valine ! • Arginine is essential for children not adults ! • Unique structural properties of many foods are derived from the various fiunctional characteristics of proteins, in conjunction with lipids and carbohydrates that are present in foods ! • Example: cheese, yogurt, tofu, etc ! !

Protein classification 1. Composition! 2. Conformation! 3. Biological function

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1. Composition • homoprotein - made up amino acids alone ! • Heteroprotein (conjugated proteins) - non protein compounds (prosthetic groups) attached to amino acids ! • Glycoprotein: k-casein (milk protein), ovalbumin (egg white protein)! • Lipoprotein: lipovitellin (egg yolk protein), plasma proteins (LDL, HDL)! • Phosphoprotein: a-casein, b-casein (milk proteins)! • Metalloprotein: hemoglobin, myoglobin (meat proteins)! • Nucleoprotein: any proteins structurally associated with nucleic acid

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2. Conformation • based on their 3D structure ! ! Globular proteins ! • 1 or more polypeptide chains fold upon themselves into spherical and globular forms ! • E.g. b-lactoglobulin, ovalbumin, myoglobin, many enzymes! • Packed tightly, compact

Fibrous proteins • • • • • ! !

composed of polypeptide chains assembled along a common straight axis, forming fibres ! E.g. collagen, keratin, elastin, actin, myosin! Long, rod like shape - twisted linear polypeptide chains! Highly ordered secondary structure (a-helix or b-sheet)! Often soluable in water !

3. Function (biological activity) Enzymes (protein catalysts)! • Protease, lipase, catalase ! ! Transport and storage proteins (ions and minerals) ! • Transferrin - transports iron into the blood ! • Hemoglobin - carries oxygen in the blood ! • Myoglobin - stores oxygen in the muscle ! ! Structural and contractile proteins ! • collagen in skin and tendon! • Keratin in hair and nail! • Actin and myosin for muscle contraction ! ! Regulatory proteins ! • control the expression of genes, the growth of body and organs, regulate metabolic reactions ! • E.g. hormones - insulin! ! Protection and defense ! • antibodies (immunoglobulins)! • Antibiotics, allergens, toxin (venom of snakeS)

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Protein building blocks: amino acids • proteins are polymers of 20 commonly occurring amino acids !

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20 amino acids • • • • • • •

all amino acids differ in their side chains (variables):! Size (molar mass)! Composition (C,O,H,S,N)! Structure (linear, ring, branched)! Polarity (polar, non polar)! Charge (neutral, positive, negative)! Hydrogen bonding capacity !

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Acid-base properties (electrical charge) of amino acids • amino acids in an aqueous solution can exist in three different ionised forms, cation +, anion -, or zwitterion, depending on the pH of the solution! • The pH at which an amino acids is electrically neutral (no net charge) is called the isoelectric point (pI)

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Electrical properties of amino acids • side groups may be neutral, positive or negative ! • Charge depends on pH relative to pKa ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Calculation of the pI amino acids

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pKa1 = pH where concentrations of COOH and COO- are equal! PKa2 = pH where concentrations of NH3+ and HN2 are equal ! Some amino acids also contain ionisable groups in side chains (pKa3)! Amino acids (no charged R groups): pI= (pKa1 + pKa2)/2! Acidic amino acids: pI = (pKa1 + pKa3)/2! Basic amino acids: pI = (pKa2 + pKa3)/2 ! In proteins, there are a limited number of ionizable groups (side chains, Nterminal amino group and C-terminal carboxyl group)

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Net charge of amino acids

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Importance of electrical properties Electrical properties are important for! • Protein structure! • Protein interactions! • Protein stability ! • Proptein functionality ! ! ! • Enables inter and intra molecules ionic interactions (electrostatic interaction or electrostatic repulsion! • Proteins may need a certain level of change in order to function ! • Many proteins are insoluable at their pI!

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Formation of peptide bond • proteins are polymers of amino acids linked together into long chains by peptide bonds ! • The formation of a peptide bond is a condensation reaction with the removal of a water molecule between the carboxyl group (COOH) of one amino acid and the amino group (NH2) of the next amino acid

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Properties of peptide bond • peptide bonds are planer and rigid and have limited rotation! • Has partial double bond characteristics! • Enable O and H to participate in H bonding between protein chains and within the same chain ! • Theoretically, ! • Torsion angles ! • Have 360 degree rotational freedom but also have limited rotation due to steric hindrance ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

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Terminologies - types of peptides • dipeptide (2 amino acids)! • Tripeptide (3 amino acids)! • Peptide (oligopeptide)! • < 50AAs, moleculer weight < 2,000 daltons ! ! Polypeptide (protein)! • a longer chain of defined sequence and length (100-10,000 AAs)! • Most proteins 100-500 AA residues ! • Manu are in the 20K - 100K daltons

Protein Structure 4 levels of structure Primary • the linear sequence of amino acids in a protein ! !

Secondary • the local 3D stricture at certain small segments of the polypeptide chain ! !

Tertiary • the global 3D conformation adopted by a single protein chain! • E.g. Globular protein, fibrous protein ! !

Quaternary • the 3D organisation of multiple protein chains

Protein structure: what determines the 3D structure • how does the protein fold?! ! Primary structure ! • amino acid type! • Amino acid number! • Amino acid sequence ! • Amino acid group bonding

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Types of tertiary structure Globular • many insoluable amino acids ! • Protein tend to minimise surface/volume ratio! !

Disordered

• if proteins do not contain secondary structures, they interact well with water and take up a random configuration ! ! ! !

Fibrous

• strong secondary structure allows protein to retain a non-spherical shape

Quartenary structure ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

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Molecular bonds involved in protein structures and protein interactions ! ! ! ! ! ! ! ! ! ! ! ! !

Molecular bonds covalent bonds • sharing of electrons between atoms ! !

Van der walls bonds • interactions between temporary dipoles ! !

Electrostatic interactions • interactions between charged groups ! !

Hydrogen bonds • strong interactions between molecule groups with strong dipoles ! • E.g. H with O, N or S! !

Hydrophobic interactions • tendency for non polar groups to associate when surrounded by water

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Protein structure, interactions and functionality depend on molecular bonds Covalent bonds • disulphide interactions !

Physical bonds • electrostatic interactions! • Van der walls interactions! • Hydrogen bonds! • Hydrophobic interactions ! ! Hydrophobic and hydrophilic properties of amino acids are also very important ! • they are major factor that affects ! • Structure and solubility of proteins ! • Surface binding properties of proteins

Protein structure: hydrophobic interactions are a major driving force favouring globular protein folding

The hydrophobic effect! • the tendency for non polar groups to associate with each other in the water ! • Due to thermodynamically unfavourable contact between non polar groups and water ! ! When there are many hydrophobic amino acids in the polypeptides —> some hydrophobic amino acids exist in patches on the surface of proteins ! ! !

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Protein Denaturation Definition • denatuation involves the transformation of a well defined folder structure of a protein, formed under physiological conditions, to an unfolded state under non physiological conditions ! ! ! ! !

! • after the secondary, tertiary or quaternary structures ! • Stabilising Forbes of protein folding such as H bond, hydrophobic interactions and ionic bonds can be broken ! ! Different conformations • there is usually one native side, but many different denatured sides !

Consequences of denaturation • the consequences of protein denaturation can be grouped into 3 aspects ! !

1. Biological 2. Chemical 3. Physical ! ! ! ! ! ! ! ! ! ! ! ! !

Biological aspects • • • • • •

enzymes lose their enzymatic activities ! Proteins exhibiting other biological activities may lose their function! E.g. antibodies, toxins, carrier and storage proteins ! Activity loss can be temporary or permanent (often irreversible)! Depending on the denaturing conditions and the length of exposure to denaturing conditions ! Some proteins will refold (renature) into their native conformation following denaturation under mild conditions

Protein denaturation: effect on enzyme activity ! ! ! ! ! ! ! ! ! Enzyme activity:! • activity depends on protein having precise molecules structure ! • Environmental conditions that significantly alter structure decrease activity ! ! ! ! ! !

Chemical aspects • unfolding of proteins due to denatuation causes reactive amino acid residues from the interior of the protein molecules to become exposed on the exterior of the molecule! • Resulting in:! • Greater reactivity of some amino acid residues ! • Possible formation of disulphide linkages and hydrophobic interactions within and between denatured proteins ! • Greater susceptibility of the peptide bond to enzymatic proteolysis ! • Thus increasing the digestibilty of proteins

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Physical aspects Native proteins are generally soluable ! • Particularly globular proteins ! • Denaturation —>! • aggregation of proteins ! • Increase in viscosity of protein solution! • Precipitation of proteins ! • Formation of protein gel! ! Common physical changes ! • It’s effect is dependant on the extent of protein denaturation ! • It’s consequence is also affected by the protein concentration! ! Denaturation often results in increased viscosity ! • the protein unfolds from a compact structure to a larger more elongated form, and thus has more resistance to flow

Globular protein denaturation and aggregation ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Denaturation: importance in foods • • • • • • • • •

denatuation plays an essential role in many food preocessing operations! Building of eggs - gelation! Cooking of meat! Baking of bread! Formation of cheese curd - loss of water solubility ! Whipping of an egg white ! Heating of a whipped egg white foams - formation of meringues ! Isolation of proteins ! Enzyme inactivation - pasteurisation of milk, blanching vegetables

Denaturing agents: physiochemical causes Important factors causing protein denatuation commonly observed with foods

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Temperature - heat denaturation • powerful denaturing ! ! Mechanism ! • weakens and disrupts non covalent interactions (H bonds, electrostatic forces, van der waals interactions) —> unfolding ! • Hydrophobic interaction is endothermic ! • This bond is relatively stable at high temperature ! • The stability of proteins against heat denaturation depends on a relative proportion of different interactions ! ! Effect on aggregation and gelation! • protein unfolding is often followed by protein aggregation! • Different disulphide linkages and hydrophobic interactions occur, resulting in the formation of a larger protein molecule! • At a protein concentration above its critical gelling level, a liquid form of the globular protein solution is converted into a semi-solid gel form ! • —> loss of solubility

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Heat denaturation When a protein solution is heated, the protein undergoes a sharp transition from the native to the denatured state above a critical temperature

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Features of thermal denaturation • • • •

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typically occurs over narrow temperature range! May be reversible or irreversible (if trapped in metastable)! May occur upon heating (heat denaturation) or cooling (cold denaturation)! Determined by balance of molecular interactions and configurational entropy !

Thermal denaturation • different proteins have different thermal denaturation temperatures depending on their structure - often practically important to know the denaturation temperature of proteins ! • The degree of thermal denaturation is also dependant on the rate of heating (temperature and time) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

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Water - affecting thermal denatuation of protein how does H20 affect thermal denatuation of protein?! Example:! Dry protein powders are very stable since in a dry state, proteins have a static structure ! When water is added, hydration and partial penetration of water into surface cavities causes swelling of the protein ! • Swelling increases chain mobility and flexibility ! • When heated, it induces greater access of H20 to salt bridges and peptide H bonds, thus lowering denatuation temperature.

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Physical denaturants: pressure Food applications of high hydrostatic pressures! • Microbial inactivation! • Protein gelation! • Meat tender is action! • Starch gelatinisation! ! Advantages over heat induced denatuation ! • does not damage essential amino acids, natural colours and flavours ! • Does not cause the formation of toxic compounds ! ! Disadvantage ! • high cost, low throughput! ! Globular protein denaturation occurs when they are placed under high hydrostatic pressures, which leads to gelation under favourable conditions ! ! ! ! ! ! ! ! ! !

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Physical denaturants: mechanical forces Shaking, kneading, whipping ! ! ! ! ! ! ! !

Mechanical forces created by shaking, kneading and whipping can cause denaturation of proteins • primarily due to the introduction of air bubbles and absorption of protein molecules to the airliquid interface ! • The air liquid interface has a surface tension greater than that of water as the air acts as a non polar phase ! • Protein molecules absorbed at interface undergo unfolding ! • Non polar amino acids residues orient towards the air; polar residues orient toward the aqueous phase ! • Principle similar, in some ways, to the protein denatuation which occurs when oils are emulsified into a protein solution

Chemical denaturants: alter molecules environment • chemical denaturants change the moleculer environment of proteins, favouring denatured (unfolded) state over native (folded) state! ! Organic solvents ! • weaken hydrophobic interactions ! ! Strong acids/bases (pH)! • alter electrostatic forces ! ! Salts, e.g. NaCl, CaCl2! • weaken hydrophobic interactions ! • Alter electrostatic forces ! ! Detergent, e.g. SDS ! • weaken hydrophobic interactions ! • Alter electrostatic forces! ! Oxidising/reducing agents! • break disulphide bonds ! • Form cross links

Chemical agent - pH • most proteins exhibit a pH range within which they are maximally stable! • At extreme pH values below 3 and above 10, most proteins are unstable and become denatured ! • This occurs as strong intramolecular electrostatic repulsion caused by high net charge results in swelling and unfolding of the protein! • Example: a high net negative or positive charge induces huge repulsive forces which lead to the protein expanding and unfolding! • This type of denaturation is often reversible ! • Some proteins are not susceptible to denaturation at extreme pH values! • Pepsin pH optimum 2 ! • Lysozyme pH optimum 11! !

Denaturation summary Factors affecting protein denaturation

Protein structure structure and nature of stabilising bonds (molecular interaction)! Presence of S-S bonds and ionic bonds in hydrophobic interior ! Tertiary structure (coil, helix, globular)! A highly ordered protein containing alpha-helix and beta sheet structures is very susceptible to thermal denaturation ! • A very thermo stable protein tends to be composed of mainly random coil

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a change in protein molecule from the native state to a non native (denatured) state ! There is usually one native state, but many different denatured states ! May be either reversible or irreversible ! Denaturation alters protein functionality and biological activity

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Functional properties of food proteins Defined as physical and chemical characteristics that impart specific functions in foods • • • • • • •

nutrition! Enzyme activity ! Solubility ! Thickening ! Gel formation ! Water holding capacity ! Emulsification!

• Foaming

Examples of functional food proteins Milk proteins ! !

Whey proteins! • multiple functions ! • Emulsification, foaming, gelation, edible filming, water holding etc ! ! ! Caseins! • curd foaming (coagulation) - cheese, yoghurt ! • Emulsification ! ! ! Egg white proteins ! • ovalbumin and lysozyme ! • Whipping properties (foaming)! • Gelation !

Wheat protein Two gluten proteins! • unique viscoelastic properties of flour dough for bread making ! • Gliadin: viscosity! • Glutenin: elasticity ! ! Meat protein! • gelatine! • Gelation ! ! ! !

Functional properties of proteins are often critical to specific food systems

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Factors determining protein functionality Molecular composition and structure ! • Conformation, charge, hydrophobicity, reactivity, interactions ! ! Environment conditions ! • pH, ionic strength, temperature ! • Solutes, solvents ! ! Goal: understand structure-function relationships

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Factors affecting functional properties of proteins

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Protein functionality: Enzyme Activity Enzyme characteristics: ! ! Enzymes are biological catalysts ! • enzymes speed up biochemical reactions without being consumed themselves ! • Enzymes are solutions to biological problems - combining molecules, breaking mol...