Ionization of Amino Acids• Amino acids contain at least two ionizable protons, eachwith its own pKa.
• The carboxylic acid has an acidic pKa and will beprotonated at an acidic (low) pH: −COOH ↔ -COO− + H+
• The amino group has a basic pKa and will be protonatedwhen basic pH (high) is achieved: −NH3+ ↔ -NH2 + H+
• At low pH, the amino acid exists in a positively charged
form (cation).
• At high pH, the amino acid exists in a negatively charged
form (anion).
• Between the pKa for each group, the amino acid exists in azwitterion form, in which a single molecule has both apositive and a negative charge.

Amino Acids Carry a Net Charge of Zeroat a Specific pH (the pI)• Zwitterions predominate at pH values between the pKavalues of the amino and carboxyl groups.
• For amino acids without ionizable side chains, theIsoelectric Point (equivalence point, pI) is:
pK1  pK 2
pI 2

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

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.
As buffers prevent change in pH close to the pKa, glycine
can act as a buffer in two pH ranges.

BufferRegions

Green Fluorescent Protein

Box 4-3a
Dr kevin Raskoff

Biologically Active Amino Acid Derivatives

Common Questions About Peptides and Proteins
What is its sequence and composition?
What is its three-dimensional structure?
How does it achieve its biochemical role?
How is its function regulated?
How does it interact with other macromolecules?
How is it related to other proteins?
Where is it localized within the cell?
What are its physico-chemical properties?

A Mixture of Proteins Can Be Separated• Separation relies on differences in physical and chemical
properties:– charge– size– affinity for a ligand– solubility– hydrophobicity– thermal stability• Chromatography is commonly used for preparative
separation in which the protein is often able to remain fully
folded.

Column Chromatography• Column chromatography
allows separation of a
mixture of proteins over asolid phase (porous matrix)
using a liquid phase tomobilize the proteins.
• Proteins with a lower
affinity for the solid phasewill wash off first; proteins
with higher affinity willretain on the column longer
and wash off later.

Separation by Charge: Ion ExchangeSeparation by Size: Size Exclusion

Separation by Binding: Affinity

Amino Acids Polymerize to Form Peptides
Amino acids → pep des → proteins• serylglycyltyrosylalanylleucine• Ser-Gly-Tyr-Ala-Leu• SGYALThe Three-Dimensional Structure of Proteins• Structure and properties of the peptide bond
• Structural hierarchy in proteins• Structure and function of fibrous proteins• Structure analysis of globular proteins• Protein folding and denaturation

Structure of Proteins• Unlike most organic polymers, protein molecules adopt a
specific three-dimensional conformation.
• 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.
• There is an entropy cost to folding the protein into one
specific native fold.

Favorable Interactions in Proteins• Hydrophobic effect– The release of water molecules from the structured solvation layer
around the molecule as protein folds increases the net entropy.

• Hydrogen bonds– Interac on of N−H and C=O of the peptide bond leads to local regularstructures such as  helices and  sheets.

• Van der Waals force– Attraction between all atoms contributes significantly to the stability inthe interior of the protein.

• Electrostatic interactions– long-range strong interactions between permanently charged groups– Salt bridges, especially those buried in the hydrophobic environment,strongly stabilize the protein.

yggfmsseks qtplvtlfkn aiiknahkkg q (31 aa)

Four Levels of Protein StructurePrimary Structure: The Peptide Bond
• The structure of the protein is partially dictated by theproperties of the peptide bond.
• The peptide bond is a resonance hybrid of two canonicalstructures.

• The resonance causes the peptide bonds:– to be less reactive compared with esters, for example– to be quite rigid and nearly planar– to exhibit a large dipole moment in the favored transconfiguration

The Rigid Peptide Plane andthe Partially Free Rotations• Rotation around the peptide bond is not permitted due toresonance structure.
• Rotation around bonds connected to the  carbon is permitted.
– f (phi): angle around the  carbon—amide nitrogen bond– y (psi): angle around the  carbon—carbonyl carbon bond
• In a fully extended polypeptide, both y and f are 180°.

The organization around the peptide bond, paired with the identityof the R groups, determines the secondary structure of the protein.

The Polypeptide Is Made Up of a Series of Planes Linked
at α Carbons

Distribution of f and y Dihedral Angles• Some f and y combinations are very unfavorable because ofsteric crowding of backbone atoms with other atoms in thebackbone or side chains.
• Some f and y combinations are more favorable because ofchance to form favorable H-bonding interactions along thebackbone.
• A Ramachandran plot shows the distribution of f and ydihedral angles that are found in a protein:• shows the common secondary structure elements• reveals regions with unusual backbone structure(-139, +135)(-51, +153)(-119, +113)(-57, -47)

Secondary Structures• Secondary structure refers to a local spatial arrangement ofthe polypeptide backbone.
• Two regular arrangements are common:• the  helix– stabilized by hydrogen bonds between nearby residues• the  sheet– stabilized by hydrogen bonds between adjacentsegments that may not be nearby• Irregular arrangement of the polypeptide chain is called therandom coil.

The  Helix• Helical backbone is held together
by hydrogen bonds between thebackbone amides of an n andn + 4 amino acids.
• It is a right-handed helix with 3.6
residues (5.4 Å) per turn.
• Peptide bonds are aligned
roughly parallel with the helicalaxis.
• Side chains point out and areroughly perpendicular with thehelical axis.

n+4nWhat Is a Right-Handed Helix?

The  Helix: Top View
• The inner diameter of the helix (no sidechains) is about 4–5 Å.
• too small for anything to fit “inside”• The outer diameter of the helix (withside chains) is 10–12 Å.
• happens to fit well into the major

groove of dsDNA• Amino acids #1 and #8 align nicely on
top of each other.

Sequence Affects Helix Stability• Not all polypeptide sequences adopt -helical structures.
• Small hydrophobic residues such as Ala and Leu arestrong helix formers.
• Pro acts as a helix breaker because the rotation aroundthe N-Ca (φ-angle) bond is impossible.
• Gly acts as a helix breaker because the tiny R groupsupports other conformations.
• Attractive or repulsive interactions between side chains 3
to 4 amino acids apart will affect formation.

The Helix Dipole• Recall that the peptide bond has a strong
dipole moment.
– C−O (carbonyl) nega ve– N−H (amide) positive• All peptide bonds in the  helix have asimilar orientation.
• The  helix has a large macroscopic
dipole moment that is enhanced byunpaired amides and carbonyls near theends of the helix.
• Negatively charged residues often occur
near the positive end of the helix dipole.

 Sheets• The planarity of the peptide bond and tetrahedral
geometry of the  carbon create a pleated sheet-likestructure.
• Sheet-like arrangement of the backbone is held together
by hydrogen bonds between the backbone amides indifferent strands.
• Side chains protrude from the sheet, alternating in anup-and-down direction.

Parallel and Antiparallel  Sheets• Multi -strand interactions are called sheets.
• Sheets are held together by the hydrogen bonding of
amide and carbonyl groups of the peptide bond fromopposite strands.
• Two major orientations of  sheets are determined by
the directionality of the strands within:– Parallel sheets have strands that are oriented in thesame direction.
– Antiparallel sheets have strands that are oriented inopposite directions.

In parallel  sheets, the H-bonded strands run in thesame direction.
• Hydrogen bonds between strands are bent (weaker).

In antiparallel  sheets, the H-bonded strands run inopposite directions.
• Hydrogen bonds between strands are linear (stronger).

 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.