Amino Acids and Proteins. 3-D Structure of Myoglobin.
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Transcript of Amino Acids and Proteins. 3-D Structure of Myoglobin.
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 polypeptide’s 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)
Leucine
Cysteine
All L-amino acids in proteins are S, except for cysteine, which is R.
Alternative Representation of Amino Acids
H3N (S) COOCH3N
CH2
COO
C=
CH3
H H3N (S) COO
=
H
CH3
H3N (R) COO
SH
CH3N
CH2
COO
SH
=H3N (R) COO
=
H SH
Properties of Cysteine Side Chain
Side chains with -SH or -OH can ionize, making them more nucleophilic.
Oxidation between pair of cysteine side chains results in disulfide bond formation.
Disulfide bonds are mainly found in extracellular proteins; the ~5 mM glutathione (-Glu-Cys-Gly) makes the inside of the cell a highly reducing environment.
oxidation
reduction
+ 2H+
+ 2e-
H3N COO
SH
H3N COO
HS
H3N COO
SS
H3N COO
H++
pKa = 8.2
H3N COO
SH
H3N COO
S
Hydroxyl-Containing Amino Acid Side Chains
Serine
Threonine
Tyrosine
H++
pKa = 13
H3N COO
OH
H3N COO
O
H++
pKa = 13
H3N COO
CH3
H3N COO
HO
H++
pKa = 10.1
H3N COO H3N COO
OH O
O CH3
Tyrosine, Serine and Threonine Can Be Phosphorylated in Proteins
Example: Tyrosine
NH
O
O
OHOH
OPO
O
O
P
O
O
OP
O
O
O N
N
N
N
NH2
O
NH
O
O
PO O
O
Tyrosyl Residue in a Protein
NH
O
O
:Base-Enzyme (Kinase)
Phosphotyrosyl Residue
+ ADP
+
Mg2+
H
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 = 3.22pI 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 (byFred 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
N-Formylmethionine inprokaryotes
Just methionine in eukaryotes
NH
HN
SCH3
O R
OO
H
H3N
HN
SCH3
O R
O
Charging of tRNAs with Specific Amino Acids
Translation of mRNA into Protein
Ribosomal Peptidyl Transferase Activity
Note: the catalytic component of the ribosome’s peptidyl transferase activity is RNA; it’s 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
Isoelectric Point (pI), pH at which molecule has net zero charge, determined using computer program for known sequence or empirically (by isoelectric focusing).
Group pKa-NH3
+ 9.7Glu -COOH 4.2Lys -NH3
+ 10.0-COOH 2.2
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
Centrifugation Methods
•Differential (Pelletting) – simple method for pelleting large particles using fixed-angle rotor (pellet at bottom of tube vs. supernatant solution above)
•Zonal ultracentrifugation (e.g., sucrose-gradient) – swinging-bucket rotor
•Equilibrium-density gradient ultracentrifugation (e.g., CsCl) – swinging-bucket or fixed-angle rotor
Low-speed, high-speed, or ultracentrifugation: different spin speeds and g forces
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
(“self-generating 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
PTH = phenylthiohydantion
F3CCOOH = trifluoroacetic acid
PTC = phenylthiocarbamyl
Identification of N-Terminal Residue
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).
NO2
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
O-I
iodoacetate
Determing Primary Structure of an Entire Protein
Reactions in Solid-Phase Peptide Synthesis