Proteins are polymers of amino acids. There are 20 unique amino acids that make up proteins in general. Each of the 20 different amino acids has a different R group. Otherwise, the amino acid structure is the same! Generic structure of an amino acid © 2014 John Wiley & Sons, Inc. All rights reserved.

KEY CONCEPTS: Section 4-1 The 20 amino acids differ in the chemical characteristics of their R groups. Amino acids are linked by peptide bonds to form a polypeptide. A protein’s structure may be described at four levels, from primary to quaternary. © 2014 John Wiley & Sons, Inc. All rights reserved.

Amino Acids: Get to Know Them!!

Amino Acids: I’m not Kidding. Make them your friends!!

© 2014 John Wiley & Sons, Inc. All rights reserved. The 20 amino acids differ in the chemical characteristics of their R groups. There are three categories for the R groups. Hydrophobic amino acids have nonpolar R groups. Hydrophilic amino acids have polar R groups. Polar R groups Nonpolar R groups Uncharged Charged © 2014 John Wiley & Sons, Inc. All rights reserved.

Glycine has a nonpolar side chain. Gly is the simplest amino acid. Side chain = H Each amino acid has: Full name (e.g., Glycine) 3 letter code (e.g., Gly) 1 letter code (e.g., G) © 2014 John Wiley & Sons, Inc. All rights reserved.

Ala and Phe have hydrophobic R groups. Ala has a methyl group for its side chain. Phe, as its name suggests, includes a phenyl ring on an Ala residue. © 2014 John Wiley & Sons, Inc. All rights reserved.

Val, Leu and Ile have hydrophobic R groups. Leu has an extra methylene group (-CH2-) than Val. Ile has the same functional moieties in its R group as Leu, just arranged differently – hence the prefix iso. V, L, and I are also referred to as Branched Chain Amino Acids (BCAAs) © 2014 John Wiley & Sons, Inc. All rights reserved.

Met has a hydrophobic R group. Met is one of only two amino acids with a sulfur in its R-group. © 2014 John Wiley & Sons, Inc. All rights reserved.

Trp has a hydrophobic R group. Trp is the only amino acid with a fused ring system. © 2014 John Wiley & Sons, Inc. All rights reserved.

Practical Application Most proteins have at least one Trp. Trp absorbs UV light at 280 nm. [Protein] can be determined based on detection of Trp in proteins! Recall Beer’s Law: Aλ = ελbc © 2014 John Wiley & Sons, Inc. All rights reserved.

© 2014 John Wiley & Sons, Inc. All rights reserved. Pro has a unique R group. Pro is the only amino acid whose side chain loops back onto its own backbone! Pro induces kinks in a polypeptide sequence. © 2014 John Wiley & Sons, Inc. All rights reserved.

Hydrophilic Amino Acids There are 2 groups of hydrophilic amino acids. Polar, uncharged Polar, charged Polar amino acids are often found in the active sites of enzymes because they can facilitate chemical catalysis. © 2014 John Wiley & Sons, Inc. All rights reserved.

Cys residues can form disulfide bonds. © 2014 John Wiley & Sons, Inc. All rights reserved.

Disulfide bonds facilitate crosslinking. Disulfide bonds can form intra-strand crosslinks (as shown). When a protein contains more than one polypeptide chain, disulfide bonds can also form inter-chain crosslinks. 3D Structure of the Protein Lysozyme Only the backbone is shown in blue. Disulfide bonds are shown in yellow ball-and-stick representation. © 2014 John Wiley & Sons, Inc. All rights reserved.

Ser and Thr have hydroxyl groups in their side chains. -OH groups are prominent nucleophiles in biochemical reactions. Other chemical groups can covalently bond to proteins via -OH groups. © 2014 John Wiley & Sons, Inc. All rights reserved.

Tyr has a polar, uncharged R group. Tyr is a derivative of Phe. Phe and Tyr are precursors of amino acid derivatives that are neurotransmitters. © 2014 John Wiley & Sons, Inc. All rights reserved.

Asn and Gln have polar, uncharged R groups. Gln has 2 methylene groups, Asn has only 1. © 2014 John Wiley & Sons, Inc. All rights reserved.

His has a polar, uncharged R group. Histidine has an imidazole (5-membered ring system with N’s). NOTE: Histidine can be charged below its pKa of ~6. © 2014 John Wiley & Sons, Inc. All rights reserved.

Lys and Arg have positively charged, polar R groups. Both Lys and Arg have long side chains with a positively charged amine group. © 2014 John Wiley & Sons, Inc. All rights reserved.

Asp and Glu have negatively charged, polar R groups. Asp and Glu are analogous to Asn and Gln, except Asp and Glu have carboxylate groups! © 2014 John Wiley & Sons, Inc. All rights reserved.

Amino acids are linked via a condensation reaction. Here amino acids within the peptide are called “amino acid residues” because only the residual atoms remain. © 2014 John Wiley & Sons, Inc. All rights reserved.

Amino acids are linked by peptide bonds to form a polypeptide Example below shows a short peptide. Polypeptides have many amino acid residues. © 2014 John Wiley & Sons, Inc. All rights reserved.

Solution: Look up the pK values for the ionization states. Recall the meaning of pK! © 2014 John Wiley & Sons, Inc. All rights reserved.

pK reveals the cutoff pH for protonation of a species. When the pH < 3.5, the structure of a generic amino acid is pK = 3.5 Both ends are protonated at very low pH. pK = 9.0 © 2014 John Wiley & Sons, Inc. All rights reserved.

pK reveals the cutoff pH for protonation of a species. When the pH > 3.5 and <9.0, the structure of a generic amino acid is Carboxyl group is deprotonated at neutral pH. Amino group is still protonated! pK = 3.5 Zwitterion Form of a Generic Amino Acid pK = 9.0 © 2014 John Wiley & Sons, Inc. All rights reserved.

pK reveals the cutoff pH for protonation of a species. When the pH > 9.0, the structure of a generic amino acid is pK = 3.5 Carboxyl group is still deprotonated. Amino group is now deprotonated! pK = 9.0 © 2014 John Wiley & Sons, Inc. All rights reserved.

© 2014 John Wiley & Sons, Inc. All rights reserved. PROBLEM: How can one deduce the structure of an amino acid or peptide when the side chain also has an ionizable group? © 2014 John Wiley & Sons, Inc. All rights reserved.

Solution: Look up the pK values for the ionization states. Recall the meaning of pK! © 2014 John Wiley & Sons, Inc. All rights reserved.

Solution: Analyze side chain pK values. EXAMPLE: His, pK = 6.0 At pH < 6.0, His side chain is protonated At pH > 6.0, His side chain is deprotonated © 2014 John Wiley & Sons, Inc. All rights reserved.

There are four different levels of protein structure. © 2014 John Wiley & Sons, Inc. All rights reserved.

KEY CONCEPTS: Section 4-2 The polypeptide backbone has limited conformational flexibility. The α helix and β sheet are common secondary structures characterized by hydrogen bonding between backbone groups. © 2014 John Wiley & Sons, Inc. All rights reserved.

Biochemists view macromolecules in a variety of ways! “Ribbon” diagram (red) Ball-and-stick atoms are superimposed Hydrogen bonds = dashed lines Space-filling representation of all atoms. Shades of blue = different subunits Shows that proteins are globular in 3D! Shows shape of backbone chain in one region of a protein is alpha helical! © 2014 John Wiley & Sons, Inc. All rights reserved.

Hydrogen Bonding in a β Sheet © 2014 John Wiley & Sons, Inc. All rights reserved.

Some polypeptide chains align in regions with directionality. Antiparallel Beta Sheets align in opposite directions. Parallel Beta Sheets align in the same direction. © 2014 John Wiley & Sons, Inc. All rights reserved.

© 2014 John Wiley & Sons, Inc. All rights reserved. Secondary Structures The α helix and β sheet are common secondary structures characterized by hydrogen bonding between backbone groups. H-bonds form in an α helix between the carbonyl oxygen and the amino hydrogen. © 2014 John Wiley & Sons, Inc. All rights reserved.

Proteins can have any combination of secondary structures. α-helical protein β-protein α/β protein Protein with very little 2° structure © 2014 John Wiley & Sons, Inc. All rights reserved.

KEY CONCEPTS: Section 4-3 A folded polypeptide assumes a shape with a hydrophilic surface and a hydrophobic core. Protein folding and protein stabilization depend on noncovalent forces. Some proteins can adopt more than one stable conformation. © 2014 John Wiley & Sons, Inc. All rights reserved.

© 2014 John Wiley & Sons, Inc. All rights reserved. A folded polypeptide assumes a shape with a hydrophilic surface and a hydrophobic core. © 2014 John Wiley & Sons, Inc. All rights reserved.

© 2014 John Wiley & Sons, Inc. All rights reserved. Protein folding and protein stabilization depend on noncovalent forces. Key example: the hydrophobic effect © 2014 John Wiley & Sons, Inc. All rights reserved.

KEY CONCEPTS: Section 4-4 Proteins containing more than one polypeptide chain have quaternary structure. © 2014 John Wiley & Sons, Inc. All rights reserved.

A single chain can form local 3D structure – domains. Domains vs. Subunits Two or more separate chains (subunits) can orient in 3D space to give quaternary structure! A single chain can form local 3D structure – domains. © 2014 John Wiley & Sons, Inc. All rights reserved.

KEY CONCEPTS: Section 4-5 Chromatography is a technique for separating molecules on the basis of size, charge, or specific binding behavior. The sequence of amino acids in a polypeptide can be determined. The 3D arrangement of atoms in a protein can be deduced by measuring the diffraction of X-rays or by analyzing NMR. © 2014 John Wiley & Sons, Inc. All rights reserved.

© 2014 John Wiley & Sons, Inc. All rights reserved. Chromatography is a technique for separating molecules on the basis of size, charge, or specific binding. Common chromatographic methods in biochemistry Gel filtration (or size exclusion) chromatography Ion exchange chromatography Affinity chromatography © 2014 John Wiley & Sons, Inc. All rights reserved.

Gel Filtration Chromatography Separation based on size Small Proteins Large Proteins © 2014 John Wiley & Sons, Inc. All rights reserved.

Ion Exchange Chromatography Separation based on charge Resins have either diethylaminoethane or carboxymethyl functional groups. © 2014 John Wiley & Sons, Inc. All rights reserved.

SDS PAGE Analysis of Proteins

Isoelectric Points of Several Common Proteins.

2D Gel Analysis - combines SDS-PAGE with Isoelectric Focusing

Crystals of the Protein Streptavidin The 3D arrangement of atoms in a protein can be deduced by measuring the diffraction of X-rays or by analyzing NMR. With X-ray crystallography, the protein must first be crystallized. © 2014 John Wiley & Sons, Inc. All rights reserved.

The NMR Structure of Glutaredoxin The 3D arrangement of atoms in a protein can be deduced by measuring the diffraction of X-rays or by analyzing NMR. With NMR spectroscopy, a family of structures is obtained. © 2014 John Wiley & Sons, Inc. All rights reserved.