Amino Acids and Proteins

3-D Structure of Myoglobin

Importance of Proteins
Main catalysts in biochemistry: enzymes (involved in virtually every biochemical reaction) Structural components of cells (both inside and outside of cells in tissues) Regulatory functions (if/when a cell divides, which genes are expressed, etc.) Carrier and transport functions (ions, small molecules)

Levels of Protein Structure

Primary Structure - amino acid sequence in a polypeptide Secondary Structure - local spatial arrangement of a polypeptides backbone atoms (without regard to side chain conformation) Tertiary Structure - three-dimensional structure of entire polypeptide Quaternary Structure - spatial arrangement of subunits of proteins composed of multiple polypeptides (protein complexes)

Structure of -amino acids

The 20 Amino Acids Found in Proteins

Properties of Different Amino Acid Side Chains

Stereochemistry of -amino acids

Stereoisomers of -amino acids

All amino acids in proteins are L-amino acids, except for glycine, which is achiral.

RS Nomenclature System (Cahn, Ingold, Prelog System)

Alternative Representation of Amino Acids

All L-amino acids in proteins are S, except for cysteine, which is R.

Leucine

CH3 H C CH3 CH2 H3N C COO H

H3N = H3N
(S)

(S)

COO

= COO

Cysteine

SH CH2 H3N C COO H

SH = H3N
(R)

= COO

H3N

(R)

COO SH

Properties of Cysteine Side Chain

pKa = 8.2 SH H3N COO H3N S + COO H+

Side chains with -SH or -OH can ionize, making them more nucleophilic.

H3N HS

COO oxidation

H3N S S

COO + 2H+ + 2e-

SH H3N COO

reduction H3N COO

Oxidation between pair of cysteine side chains results in disulfide bond formation.

Disulfide bonds are mainly found in extracellular proteins; the ~5 mM glutathione (g-Glu-Cys-Gly) makes the inside of the cell a highly reducing environment.

Hydroxyl-Containing Amino Acid Side Chains

pKa = 13 OH O + H+ H3N COO

Serine

H3N

COO

pKa = 13 HO CH3 COO O H3N CH3 + H3N COO H+

Threonine

OH

pKa = 10.1

O + H+

Tyrosine
H3N COO H3N COO

Tyrosine, Serine and Threonine Can Be Phosphorylated in Proteins

Example: Tyrosine
O H :Base-Enzyme (Kinase)

N H

Tyrosyl Residue in a Protein

O NH2 O O O O O P O P O P O O O O Mg2+ N N O OH OH N N H N P O

O O

+ N H

Phosphotyrosyl Residue + ADP

Modified or Unusual Amino Acids

Absorption of UV Light by Aromatic Amino Acids

Titration of Amino Acids with Ionizing Side Chains

Isoelectric point (pI) for amino acids with ionizable side chains: Take average pKa for the two ionizations involving the neutral (net charge of zero) species. pI of Glu = (2.19 + 4.25)/2 = 3.22 pI of His = (6.0 + 9.17)/2 = 7.59

Formation of a Peptide

Planarity of Peptide (Amide) Bond

cis and trans Isomers

The trans isomer is generally more stable because of steric crowding of side chains in the cis isomer.

Examples of Oligopeptides

N- and C-Termini May Be Modified in Proteins

Primary Structure of Bovine Insulin

First protein to be fully sequenced (by Fred Sanger in 1953). For this, he won his first Nobel Prize (his second was for the Sanger dideoxy method of DNA sequencing).

Evolution and Conservation of Protein Sequences

Translation elongation factor Tu/1

Myoglobin

The Genetic Code

DNA

RNA

Protein

Initiating Amino Acid in Translation

CH3 S O H N H H N O R O

N-Formylmethionine in prokaryotes

CH3 S H N O R O

H 3N

Just methionine in eukaryotes

Charging of tRNAs with Specific Amino Acids

Translation of mRNA into Protein

Ribosomal Peptidyl Transferase Activity

Note: the catalytic component of the ribosomes peptidyl transferase activity is RNA; its an example of a catalytic RNA or ribozyme.

Disulfide Bond Formation in Insulin

Methods in Protein Biochemistry

Gel Electrophoresis

Polyampholyte Character of a Tetrapeptide and Isoelectric Points

Group pKa -NH3+ 9.7 Glu g-COOH 4.2 Lys e-NH3+ 10.0 -COOH 2.2

Isoelectric Point (pI), pH at which molecule has net zero charge, determined using computer program for known sequence or empirically (by isoelectric focusing).

Isoelectric Focusing
Electrophoresis through polyacrylamide gel in which there is a pH gradient.

Two-Dimensional Gel Electrophoresis

Separate proteins based on isolectric point in 1st dimension Separate proteins based on molecular weight in 2nd dimension

Salting Out: Ammonium Sulfate Precipitation in Protein Fractionation

Centrifugation
Low-speed, high-speed, or ultracentrifugation: different spin speeds and g forces

Centrifugation Methods
Differential (Pelletting) simple method for pelleting large particles using fixedangle rotor (pellet at bottom of tube vs. supernatant solution above) Zonal ultracentrifugation (e.g., sucrosegradient) swinging-bucket rotor

Equilibrium-density gradient ultracentrifugation (e.g., CsCl) swinging-bucket or fixed-angle rotor

Zonal Centrifugation: Sucrose-Gradient Preparative Ultracentrifugation

Separates by sedimentation coefficient (determined by size and shape of solutes)

Sucrose-Gradient Preparative Ultracentrifugation

Equilibrium Density Gradient Ultracentrifugation

Used in Meselsen-Stahl experiment. Separates based on densities of solutes. Does not require premade gradient. Pour dense solution of rapidly diffusing substance in tube (usually CsCl). Density gradient forms during centrifugation (selfgenerating gradient). Solutes migrate according to their buoyant density (where density of solute = density of CsCl solution).

Column Chromatography

Flow-through

Eluate

Different Types of Chromatography

Gel filtration/size exclusion/molecular sieve - separates by size (molecular weight) of proteins Ion exchange (cation exchange and anion exchange) separates by surface charge on proteins
Cation exchange: separates based on positive charges of solutes/proteins, matrix is negatively charged Anion exchange: separates based on negative charges of solutes/proteins, matrix is positively charged

Hydrophobic interaction - separates by hydrophobicity of proteins Affinity - separates by some unique binding characteristic of protein of interest for affinity matrix in column

Ion-Exchange Chromatography

Gel Filtration Chromatography

Affinity Chromatography

Cleavage of Polypeptides for Analysis

Strong acid (e.g., 6 M HCl) - not sequence specific Sequence-specific proteolytic enzymes (proteases) Sequence-specific chemical cleavage (e.g., cyanogen bromide cleavage at methionine residues)

Protease Specificities

Cyanogen Bromide Cleavage at Methionine Residues

Protein Sequencing: Edman Degradation

PTC = phenylthiocarbamyl F3CCOOH = trifluoroacetic acid

PTH = phenylthiohydantion

Identification of N-Terminal Residue

NO2

Note: Identification of C-terminal residue done by hydrazinolysis (reaction with anhydrous hydrazine in presence of mildly acidic ion exchange resin) or with a C-terminus-specific exopeptidase (carboxypeptidase).

Separation of Amino Acids by HPLC

Protein Identification by Mass Spectrometry

Protein Identification by Mass Spectrometry

Two main approaches: 1. Peptide mass fingerprinting: Proteolytic digestion of protein, then determination m/z of peptides by MS (e.g., MALDI-TOF or ESI-TOF), search fingerprint against database. Success of ID depends on quality/ completeness of database for specific proteome. 2. Tandem MS (MS/MS e.g., nanoLC-ESI-MS/MS): Proteolytic digestion of protein, separation and determination of m/z of each (MS-1), then determination of collision-induced dissociation fragment spectrum for each peptide (MS-2). Gives context/sequence-dependent information, so more of a do novo sequencing method.

Locating Disulfide Bonds

O I Oiodoacetate

Determing Primary Structure of an Entire Protein

Reactions in Solid-Phase Peptide Synthesis