CH.5: Peptides & Proteins

Proteins

• Are an essential part of all organisms

• Make about ½ of the dry weight of the human body

• Have many biological functions

• Their functional diversity is due to the variety of sizes & shapes that they can assume

Enzymes

A protein that serves as a biological catalyst & it speeds up biochemical reactions

Antibodies

Immunoglobulins are the body’s defense mechanism that is produced by the immune system in response to foreign antigens like viruses & bacteria. They bind to the foreign antigen to help destroy or remove it.

• Fibrinogen & Fibrin are proteins that are involved in blood clotting (this serves as a protection over an open wound)

Fibrinogen & Fibrin are…

…Proteins that are involved in blood clotting (this serves as a protection over an open wound)

Regulator Proteins

These control many cell functions such as metabolism & reproduction. The hormone insulin & glucagon regulate blood glucose. Transcription factors control gene expression.

Structural Proteins

These provide large animals with mechanical support. Collagen provide mechanical strengths to bones, tendons & skin. Elastin are found in blood vessels & ligaments.

Contractile Proteins

The interaction of actin & myosin proteins are responsible for the contraction & expansion of muscles & the heart.

Nutrient Proteins

Egg albumin & casein in milk are nutrient storage proteins. They serve as sources of amino acids for embryos & infants

What is a protein?

Proteins are polymers that are composed of different alpha-amino acids that are linked together through peptide bonds.

• Protein and Polypeptide can be used interchangeably

Peptides contain…

…Between 2 & 50 amino acids

Proteins contain…

…More than 50 amino acids

What does the structure, function & activity of a protein are determined by?

They are determined to a large extent by the number, sequence & chemical properties of its amino acids

Amino Acids

• Basic structural unit of proteins

• There are over 100 different amino acids in living organisms

• Only 20 amino acids are coded by DNA & are commonly found in protein. These are referred to as standard amino acids

• The nonstandard amino acids are produced by modification of one of the original 20 amino acids after it has been incorporated into a polypeptide

Standard Amino Acids

Only 20 amino acids are coded by DNA & are commonly found in protein

Nonstandard Amino Acids

Are produced by the modification of one of the original 20 amino acids after it has been incorporated into a polypeptide

General Structure of Amino Acids

In the cell & in aqueous solution:

• The carboxylic acid exists as a carboxylate anion. The amino group is protonated to NH₃⁺. Hence, it exists as a dipolar ion (zwitterion)

• 19 of 20 standard amino acids have this type of general structure

• Only 1, proline, is an imino acid as the side chain is directly bonded to the amino group

• The amino acids are differentiated by their side chains

Stereochemistry of Amino Acids

• The alpha carbon of all amino acids except glycine is chiral. Therefore, all except glycine exist as pairs of enantiomers

• The enantiomers are of the L- & D-isomers: amino group is to the left of the chiral carbon in the L-isomer & the amino group is to the right of the chiral carbon in the D-isomer

• The alpha amino acids from proteins are L-isomer configurations (this is their natural form)

Classes of Amino Acids

Amino acids have been divided into categories based on the similarities & differences in their side chains

• Nonpolar Neutral

• Polar Neutral

• Polar Acidic (negative)

• Polar Basic (positive)

Nonpolar Neutral

Contain hydrocarbon (nonpolar) R groups. These are hydrophobic. Neutral because the net charge is 0/

• Ala, Val, Leu, Ile, Pro, Gly, Met, Cys, Phe & Trp

Polar Neutral

Contain polar non-ionic R groups that are capable of hydrogen bonding. These are hydrophilic amino acids

• Ser, Thr, Tyr, Asn & Gln

Polar Acidic (negative)

R groups have ionized carboxyl groups. At pH 7, these amino acids have a net charge of -1. These are hydrophilic amino acids

• Asp & Glu

Polar Basic (positive)

R groups contain a positive group. Net charge is +1. These are hydrophilic

• His, Arg & Lys

Ionic State of Amino Acids

• Free amino acids have at least 2 ionizable groups

• At pH 7, the -COOH group is in its conjugate base form (COO^-) & the -NH₂ group is in its conjugate acid form (NH₃⁺), The net charge is 0

• Amino acids are amphoteric because they can act as an acid or as a base

• The exact charge on an amino acid depends on the pK_a’s of the amino group, carboxyl group, side chain ionizable group (if any) & the pH of the solution

• The pK_a of amino group is ~9-10 & the carboxyl group is 1.7-2.4

Amphoteric

Amino acids are this because they can act as an acid or as a base

Isoelectric Point (pI)

Is the pH at which an amino acid has no net charge & is electrically neutral

Where is the isoelectric point for amino acids that contain no ionizable group on its side chain?

The isoelectric point is midway between the pK_a’s of the carboxyl & the amino groups

The amino acids with ionizable side chains have…

…More complex titration curves. Isoelectric point is the average of the two pK_a’s nearest to it (or flanking)

pH < pI

At a pH below its pI, an amino acid or protein carries a net positive charge & will migrate towards a negative electrode (cathode)

pH > pI

At a pH above its pI, an amino acid or protein carries a net negative charge & will migrate towards a positive electrode (anode)

• Note: the pK_a values for amino acids in protein & peptides differ somewhat from the values of free amino acids

Dipeptide

When two amino acids link, it forms this (Alanine & Serine can form Alanylserine or Serylalanine)

Tripeptide

Contains 3 amino acids linking together

Tetrapeptide

Contains 4 amino acids linking together

N-Terminal residue

Is the residue with the free amino group & is written on the left

C-Terminal residue

Has the free carboxyl group on it & appears on the right

Biological Function of Peptides

Naturally occurring peptides are responsible for a number of biological functions:

• Glutathione

• Oxytocin

• Vasopressin

• Met-enkephalin & Leu-enkephalin

Glutathione

A tripeptide that protects the cell from oxidation

Oxytocin

A 9 amino acid peptide that induces labor

Vasopressin

Differs from oxytocin by 2 amino acids. Regulates blood pressure & the kidney in order to retain water

Met-enkephalin & Leu-enkephalin

Neuropeptide hormones that serve as a pain blockage & also inhibit intestinal motility & blood flow to the gastrointestinal tract. The action of enkephalin are short-lived.

Protein Classification

Proteins may be classified according to chape & composition

• Shape

  • Fibrous proteins

  • Globular proteins

• Composition

  • Simple proteins

  • Conjugated proteins

Nonprotein components—prosthetic group

Proteins without prosthetic group—apoprotein

Protein plus prosthetic group—holoprotein

Protein Classification—Shape

• Fibrous proteins

• Globular proteins

Fibrous Proteins

Long, rod shaped structure. Insoluble in water & physically tough. Have structural & protective functions.

Globular Proteins

Compact, spherical proteins that are tightly folded together. Water soluble & serve diverse functions.

Protein Classification—Composition

• Simple proteins

• Conjugated proteins

Simple Proteins

Contain only amino acids

Conjugated Proteins

Contain amino acids & a nonprotein component

Functional protein with a prosthetic group

• Are classified according to the nature of their prosthetic groups

  • Glycoproteins

  • Lipoproteins

  • Phosphoproteins

  • Metaloproteins

  • Hemoproteins

Glycoproteins

Contain carbohydrate component

Lipoproteins

Contain lipid molecules

Phosphoproteins

Contain phosphate prosthetic group

Metaloproteins

Contain metal ion

Hemoproteins

Heme prosthetic group

Prosthetic Group

Nonprotein component

Apoprotein

Protein without prosthetic group

Holoprotein

Protein plus prosthetic group

Protein Structure

• Primary structure

• Secondary structure

• Tertiary structure

• Quaternary structure

Primary Structure

The amino acid sequence specified by genetic information. The primary sequence also contains information to determine how it will fold & function

Secondary Structure

Formed by the folding of a primary structure into a regularly repeated structure as a result of H bonding between the amide hydrogen & the carbonyl oxygen. Most common types are the alpha helix & the beta pleated sheet

Structure of Secondary Structure

• Alpha helix

• Rigid, rodlike right-handed coiled structure

• Held together by H bonds between the amide hydrogen of residue n and the carbonyl oxygen of residue (n + 4)

• H bonds are parallel to the long axis of the helix

• 3.6 residues/turn

• Side chains point away from the interior of the helix

• Amino acids that favor alpha helix formation includes: Ala, Leu & Met

• Amino acids that disrupt alpha helix includes: Pro, Gly & Trp

• Strands of alpha helix are held together by cross-linking cystine residue

Tertiary Structure

Three dimensional structure where the peptide chain containing the secondary structures further fold on itself. These are globular proteins. The forces that hold these protein structure includes:

• Hydrogen bonds for polar amino acids

• Ionic bridges between oppositely charged amino acids

• Van der Waals forces dipole-dipole type interactions

• Hydrophobic interactions between nonpolar amino acids

• Covalent bonds formed by the disulfide bridges

Quaternary Structure

The association of several globular peptides to produce a functional protein. Forces that hold these structures are the same as tertiary structure. Each peptide component in the protein complex is a subunit. The subunits may be identical or different.

Advantages of Multisubunit Proteins:

• More efficient to synthesize separate subunits than to increase the length of a single polypeptide substantially

• Replacement of smaller, worn-out or damaged components is easier

• The complex interactions of multiple subunits regulate function

Beta-Pleated Sheet

• Two or more polypeptide chains line up in an extended zigzag structure.

• R groups alternate above & below the plane of polypeptide chains.

• There are no intrachain H bonds but interchain H bonds between the carbonyl oxygen of one chain & the amide hydrogen of an adjacent chain.

• There are two beta-pleated sheets: parallel & antiparallel

• There are no cystine cross-linkages between their side chains.

• Beta-pleated sheets are only formed in polypeptides that contain small side chains such as: Ala, Gly & Ser

Parallel

N to C terminal direction of adjacent chains are the same

Antiparallel

N to C terminal direction of adjacent chains are the opposite

Fibrous Protein Structure

• Proteins arranged in fibers or sheets

• Contain a high proportion of secondary structure (alpha helix or beta pleated sheet)

• The 4 kinds of fibrous proteins are:

  • Alpha keratin

  • Beta keratin

  • Collagen

  • Elastin

What are the four kinds of fibrous proteins?

• Alpha keratin

• Beta keratin

• Collagen

• Elastin

Alpha keratins

It has the alpha helix structure. High cystine content, which provides disulfide bonds between adjacent chains. They are insoluble protective structures of great mechanical strength, varying in hardness & flexibility. (E.g. hair, wool, nails, hooves, fur & tortoise shell)

Beta keratins

Fibrous proteins are composed of beta pleated sheets. (E.g., silk fibroin & spider webs). Both have very high content of glycine & alanine. In silk, the alternate amino acid is glycine. The structure of silk fibroin is an antiparallel beta pleated sheet. Silk does not stretch easily. They are soft & flexible.

Collagen

• It is the most abundant protein in the body

• Is found in tendons, skin fibers, blood vessels, bone & cartilage

• Their fibrils consist of recurring polypeptide subunits called troponocollagen

  • These fibrils do not stretch & have great tensile strength

• Contains a high proportion of glycine (35%), alanine (11%), proline & 4-hydroxyproline (30%) & a small amount of 3-hydroxyproline & 5-hydroxylysine

• The amino acid consists of a repeating triplet sequence of Gly-X-Y (X is often proline & Y is 4-hydroxyproline or 5-hydroxylysine)

• Is converted into gelatin by partial hydrolysis of some of the covalent bonds of collagen

Tropocollagen

Recurring polypeptide subunits arranged head-to-tail in parallel bundles that are within collagen fibrils

• Consists of 3 left-handed polypeptide helices wrapped around one another in the right-handed sense

  • The 3 helices are held together by hydrogen bonds & by an unusual covalent cross-link formed between lysine residues on the adjacent chain

The Role of Vitamin C

• Hydroxyproline & hydroxylysine are formed by post-translational modification. Collagen protein is synthesized with proline & lysine in the amino acid chain. The enzyme prolyl hydroxylase then hydroxylate proline to 4-hydroxyproline & lysyl hydroxylase then hydroxylate lysine to 5-hydroxylysine

• Both enzymes require vitamin C to carry out the hydroxylation reactions

• Lack of vitamin C causes scurvy which is characterized by skin lesions, fragile blood vessels & bleeding gums

Elastin

• Ligaments are rich in elastin

• Their fibrils are flexible & elastic

• Consists of a network of polypeptide chains cross-linked by desmosine (a derivative of lysine found only in elastin)

Globular Protein Structure

• Protein that folds into a well-defined 3-D structure

• Extremely compact

• Some must bind to a prosthetic group in order to be functional (e.g., heme group of Hb & Mb)

• A functional protein with a prosthetic group is called a conjugated protein

Myoglobin

• (Mb) is the oxygen storage protein of muscle

• It has a higher affinity for oxygen than hemoglobin

• High concentration in diving animals

• It is a relatively small protein-153 amino acids

• The protein component (globin) is a single polypeptide chain that contains 8 sections of an alpha helix

• Heme prosthetic group which binds oxygen is located within a hydrophobic crevice

Hemoglobin

• (Hb) is the oxygen transport protein found in red blood cells

• It is composed of 4 peptide subunits: 2 alpha & 2 beta subunits

• The 4 subunits of hB are arranged in 2 identical dimers: alpha 1 beta 1 & alpha 2 beta 2

• Each subunit contains a heme group & the Fe²⁺ ion binds the oxygen. Therefore, a Hb molecule can bind 4 oxygen molecules

• Hb has two conformations: the deoxyhemoglobin is in the T (taut) state. The oxyhemoglobin is in the R (related) state

O₂ Binding Curve

• Binding of oxygen to hemoglobin is cooperative

• The binding of the first O₂ facilitates the binding of the remaining 3 O₂ to the Hb molecule

• The oxygen dissociation curve of Hb is sigmoidal shape because of the interactions between the subunits

• The oxygen dissociation curve of Mb is hyperbolic shape as Mb has a greater affinity for oxygen than Hb & the curve is well to the left of Hb curve

What is the oxygen dissociation curve of Hb?

Sigmoidal shape because of the interactions between the subunits

What is the oxygen dissociation curve of Mb?

Hyperbolic shape because it has a greater affinity for oxygen than Hb & the curve is well to the left of Hb curve

Oxygen Binding Affinity

Factors that affect/facilitate hemoglobin binding oxygen:

• The binding of the first oxygen to hemoglobin changes

  • deoxyHb(taut)—>oxyHb(relaxed)

    • The relaxed form has a high affinity to bind oxygen

• The 2,3-biophosphoglycerate, which stabilizes the deoxyhemoglobin in the taut form, is expelled. This keeps the Hb in the relaxed form, which has a higher affinity for oxygen

• The release of H⁺ from hemoglobin into the blood. H⁺ interacts with ionizable groups on Hb & stabilizes it in the taut form. Its release keeps Hb in the relaxed form, which has a higher affinity for oxygen

Hb & Mb Function (in the lung)

• O₂ diffuses from high partial pressure (100mmHg pO₂) to low pO₂ in the oxygen depleted blood

• O₂ enters the red blood cells & binds to Fe²⁺ ions of the heme group of deoxyHb (taut state) forming oxyHb (relaxed state)

• The change in shape causes the organic anion 2,3-biophosphoglycerate (BPG) which binds in the center of Hb to be expelled. This speeds up the ability of the remaining 3 subunits to accept O₂ because in the absence of BPG hemoglobin has a very high affinity for O₂

• When deoxyHb binds oxygen, it releases protons which are removed by the enzyme carbonic anhydrase in the reaction:

  • H⁺ + HCO₃^- —> CO₂ + H₂O

• The HCO₃^- originally formed by the reverse reaction when CO₂ from actively metabolizing tissue enters the blood. The removal of H⁺ from the blood stimulates deoxyHb to bind O₂

Hb & Mb Function (at the tissue)

• When oxygenated blood reaches actively metabolizing tissues, waste CO₂ diffuses into the blood & enters the red blood cells where carbonic anhydrase catalyses the reverse reaction:

  • CO₂ + H₂O —> H⁺ + HCO₃^-

• The increase in H+ in the blood binds to several ionizable groups on the Hb, causing the release of oxygen from oxyHb by converting it to the deoxyHb conformation. As soon as oxygen is released from 1 Hb subunit, BPG begins to work its way back to the space in the center of the hemoglobin molecule. This stabilizes the deoxyHb (taut form), causing the release of oxygen quickly. Since myoglobin has a higher affinity for O₂, it quickly binds oxygen that diffuses from the higher pO₂ in the blood to the tissue

Protein Denaturation

• Disruption of the native conformation of a protein along with loss of biological activity

• Generally referred to as disruption of tertiary structure, not breaking of peptide bonds

• May be reversible or irreversible

• Denaturing conditions:

  • Extreme pH change

  • Organic solvents

  • Detergents

  • Reducing agents

  • Salt concentration

  • Heavy metals

  • Extreme temperature changes

  • Mechanical stress

Extreme pH change

Result in protonation of the protein side groups, which disrupted H bonds & ionic interaction. Protein unfolds, coagulates & precipitates out of solution

Organic solvents

(Such as Ethanol) interferes with the hydrophobic interaction of the nonpolar R groups by forming H bonds with water & the polar groups. Nonpolar solvents can also disrupt hydrophobic interactions

Detergents

Disrupt hydrophobic interactions

Reducing agents

(Such as beta-mercaptoethanol) breaks disulfide bonds by reducing them to sulfhydryl groups

Salt concentration

Small amounts of salt can improve solubility (“salting-in”). Salt ions bind to the protein’s ionizable groups, decreasing charged-group interactions & water can form solvated spheres around these groups. Large amounts of salt compete with the protein for the water molecules surrounding the ionized groups, causing the protein to precipitate (“salting-out”)

Heavy metals

(Such as mercury & lead) can disrupt salt bridges & sulfhydryl groups

Extreme temperature changes

Disrupt H bonds which causes the protein to unfold, becoming entangled & coagulate

Mechanical stress

(Such as whipping & shaking) can disrupt a variety of forces that maintain the protein structure