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Amino Acids, Peptides, Proteins

Structure and naming of amino acids Structure and properties of peptides Ionization behavior of amino acids and peptides Methods to characterize peptides and proteins

Proteins: Main Agents of Biological Function

Catalysis:
enolase (in the glycolytic pathway) DNA polymerase (in DNA replication)

Transport:
hemoglobin (transports O2 in the blood) lactose permease (transports lactose across the cell membrane)

Structure:
collagen (connective tissue) keratin (hair, nails, feathers, horns)

Motion:
myosin (muscle tissue) actin (muscle tissue, cell motility)

Amino Acids: Building Blocks of Protein

Proteins are heteropolymers of -amino acids Amino acids have properties that are well suited to carry out a variety of biological functions:
Capacity to polymerize Useful acid-base properties Varied physical properties Varied chemical functionality

General structure of an amino acid

Amino Acids: Atom Naming

Organic nomenclature: start from one end Biochemical designation: start from -carbon and go down the R-group

Most -Amino Acids are Chiral

The -carbon has always four substituents and is tetrahedral All (except proline) have an acidic carboxyl group, a basic amino group, and an alpha hydrogen connected to the -carbon Each amino acid has a fourth substituent R In glycine, the fourth substituent is also hydrogen

Amino Acids: Classification

Common amino acids can be placed in five basic groups depending on their R substituents: Nonpolar, aliphatic (7) Aromatic (3) Polar, uncharged (5) Positively charged (3) Negatively charged (2)

These amino acid side chains are hydrophobic

These amino acid side chains absorb UV light at 270-280 nm

These amino acids side chains can form hydrogen bonding Cysteine can form disulfide bonds

Uncommon Amino Acids in Proteins

Not incorporated by ribosomes Arise by post-translational modifications of proteins

Reversible modifications, esp. phosphorylation is important in regulation and signaling

Ionization of Amino Acids

At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions At alkaline pH, the amino group is neutral NH2 and the amino acid is in the anionic form.

Chemical Environment Affects pKa Values

-carboxy group is much more acidic than in carboxylic acids -amino group is slightly less basic than in amines

The stronger the tendency to dissociate a proton, the stronger is the acid and the lower its pKa.

Amino Acids Can Act as Buffers

Amino acids with uncharged side-chains, such as glycine, have two pKa values: - The pKa of the -carboxyl group is 2.34 - The pKa of the -amino group is 9.6 It can act as a buffer in two pH regimes.

Amino Acids Carry a Net Charge of Zero at a Specific pH

Zwitterions predominate at pH values between the pKa values of amino and carboxyl group For amino acid without ionizable side chains, the Isoelectric Point (equivalence point, pI) is

pI =

pK + pK 2
1

At this point, the net charge is zero AA is least soluble in water AA does not migrate in electric field

Ionizable Side Chains Can Show Up in Titration Curves

Ionizable side chains can be also titrated Titration curves are now more complex pKa values are discernable if two pKa values are more than two pH units apart Why is the side-chain pKa so much higher?

How to Calculate the pI When the Side-chain is Ionizable?

Identify species that carries a net zero charge Identify pKa value that defines the acid strength of this zwitterion: (pK2) Identify pKa value that defines the base strength of this zwitterion: (pKR) Take the average of these two pKa values

Formation of Peptides
Peptides are small condensation products of amino acids They are small compared to proteins (Mw < 10 kDa)

Peptide Ends are Not the Same

AA1 AA2 AA3 AA4 AA5

Numbering starts from the amino terminus

The Three Letter Code

Naming starts from the N-terminus Sequence is written as: Ala-Glu-Gly-Lys Sometimes the one-letter code is used: AEGK

Peptides: A Variety of Functions

Hormones and pheromones:
insulin (think sugar) oxytocin (think childbirth) sex-peptide (think fruit fly mating)

Neuropeptides
substance P (pain mediator)

Antibiotics:
polymyxin B (for Gram - bacteria) bacitracin (for Gram + bacteria)

Protection, e.g. toxins

amanitin (mushrooms) conotoxin (cone snails) chlorotoxin (scorpions)

Proteins are:
Polypeptides (covalently linked -amino acids) and
possibly cofactors, coenzymes, prosthetic groups, other modifications Cofactor is a general term for functional non-amino acid component
Metal ions or organic molecules

Coenzyme is used to designate an organic cofactors

NAD+ in lactate dehydrogenase

Prosthetic groups are covalently attached cofactors

Heme in myoglobin

Polypeptide Size in Some Proteins

Classes of Conjugated Proteins

The Study of Peptides and Proteins

The sequence and composition The three-dimensional structure The native fold The biochemical role Function regulation Interaction with other macromolecules Relation to other proteins Localization within the cell The physical and chemical properties

A Mixture of Proteins Can Be Separated

Separation relies on differences in physicochemical properties
Charge Size Affinity for a ligand Solubility Hydrophobicity Thermal stability

Chromatography is commonly used for preparative separation

Electrophoresis for Protein Analysis

Separation in analytical scale is commonly done by electrophoresis
Electric field pulls proteins according to their charge Gel matrix hinders mobility of proteins according to their size and shape

SDS PAGE: Molecular Weight

SDS sodium dodecyl sulfate a detergent

SDS micelles binds to, and unfold all the proteins

SDS gives all proteins an uniformly negative charge The native shape of proteins does not matter Rate of movement will only depend on size: small proteins will move faster

SDS Gel Electrophoresis

Protein Sequencing

Spectroscopic Detection of Aromatic Amino Acids

The aromatic amino acids absorb light in the UV region Proteins typically have UV absorbance maxima around 275-280 nm Tryptophan and tyrosine are the strongest chromophores Concentration can be determined by UV-visible spectrophotometry using Beers law:

A = cl

Summary
The many biological functions of peptides and proteins The structures and names of amino acids found in proteins The ionization properties of amino acids and peptides The methods for separation and analysis of proteins

Proteins: Structure, Function, Folding

Structure and properties of the peptide bond Structural hierarchy in proteins Structure and function of fibrous proteins Structure analysis of globular proteins

Structure of Proteins
Unlike most organic polymers, protein molecules adopt a specific 3-dimensional conformation in the aqueous solution. This structure is able to fulfill a specific biological function This structure is called the native fold The native fold has a large number of favorable interactions within the protein

Favorable Interactions in Proteins

Hydrophobic effect
Release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy

Hydrogen bonds
Interaction of N-H and C=O of the peptide bond leads to local regular structures such as -helixes and -sheets

London dispersion
Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein

Electrostatic interactions
Long-range strong interactions between permanently charged groups Salt-bridges, esp. buried in the hydrophobic environment strongly stabilize the protein

Structure of the Peptide Bond

Structure of the protein is partially dictated by the properties of the peptide bond The peptide bond is a resonance hybrid of two canonical structures The resonance causes the peptide bonds be less reactive compared to e.g. esters be quite rigid and nearly planar exhibit large dipole moment in the favored trans configuration

The Rigid Peptide Plane and the Partially Free Rotations

Rotation around the peptide bond is not permitted Rotation around bonds connected to the alpha carbon is permitted (phi): angle around the -carbonamide nitrogen bond (psi): angle around the -carboncarbonyl carbon bond In a fully extended polypeptide, both and are 180

Distribution of and Dihedral Angles

Some and combinations are very unfavorable because of steric crowding of backbone atoms with other atoms in the backbone or side-chains Some and combinations are more favorable because of chance to form favorable H-bonding interactions along the backbone Ramachandran plot shows the distribution of and dihedral angles that are found in a protein shows the common secondary structure elements reveals regions with unusual backbone structure

Secondary Structures
Secondary structure refers to a local spatial arrangement of the polypeptide chain Two regular arrangements are common: The helix
stabilized by hydrogen bonds between nearby residues

The sheet
stabilized by hydrogen bonds between adjacent segments that may not be nearby

Irregular arrangement of the polypeptide chain is called the random coil

The helix
The backbone is more compact with the dihedral (N CCN) in the range ( 0 < < 70) Helical backbone is held together by hydrogen bonds between the nearby backbone amides Right-handed helix with 3.6 residues (5.4 ) per turn Peptide bonds are aligned roughly parallel with the helical axis Side chains point out and are roughly perpendicular with the helical axis

Sequence Affects Helix Stability

Not all polypeptide sequences adopt -helical structures Small hydrophobic residues such as Ala and Leu are strong helix formers Pro acts as a helix breaker because the rotation around the N-Ca bond is impossible Gly acts as a helix breaker because the tiny Rgroup supports other conformations

 Sheets
The backbone is more extended with the dihedral (NCCN) in the range ( 90 < < 180) The planarity of the peptide bond and tetrahedral geometry of the -carbon create a pleated sheetlike structure Sheet-like arrangement of backbone is held together by hydrogen bonds between the more distal backbone amides Side chains protrude from the sheet alternating in up and down direction

Parallel and Antiparallel Sheets

Parallel or antiparallel orientation of two chains within a sheet are possible In parallel sheets the H-bonded strands run in the same direction In antiparallel sheets the H-bonded strands run in opposite directions

 Turns
-turns occur frequently whenever strands in sheets change the direction The 180 turn is accomplished over four amino acids The turn is stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence Proline in position 2 or glycine in position 3 are common in -turns

Protein Tertiary Structure

Tertiary structure refers to the overall spatial arrangement of atoms in a polypeptide chain or in a protein One can distinguish two major classes fibrous proteins- long strands or sheets typically insoluble; made from a single secondary structure globular proteins- spherical or globular shape water-soluble globular proteins lipid-soluble membrane proteins

Tertiary structure of sperm whale myoglobin

Structure of collagen

Quaternary Structure

Quaternary structure is formed by spontaneous assembly of individual polypeptides into a larger functional cluster

Quaternary structure of deoxyhemoglobin

Problem
11. Net Electric Charge of Peptides A peptide has the sequence GluHisTrpSerGlyLeuArgProGly (a) What is the net charge of the molecule at pH 3, 8, and 11? (Use pKa values for side chains and terminal amino and carboxyl groups as given in Table 31.) (b) Estimate the pI for this peptide.

(a) When pH >pKa, ionizing groups lose their protons. GluHisTrpSerGlyLeuArgProGly

(b) Find the point at which net peptide charge 0 (here, two groups that ionize near pH 8): the amino-terminal a-amino group of Glu and the His imidazole group.