Exam 3 Biochem Flashcards
Main Agents of Protein Biological Function
Catalysis
Transport
Structure
Motion
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
Amino Acids: Classification
nonpolar, aliphatic (7)
aromatic (3)
polar, uncharged (5)
positively charged (3)
negatively charged (2)
nonpolar, aliphatic (7)
glycine
alanine
proline
valine
leucine
isoleucine
methionine
aromatic (3)
phenylalanine
tyrosine
tryptophan
polar, uncharged (5)
serine
threonine
cysteine
asparagine
glutamine
positively charged (3)
lysine
arginine
histidine
negatively charged (2)
aspartate
glutamate
Ionization of Amino Acids
contain at least two ionizable protons, each with its own pKa.
The carboxylic acid has an acidic pKa and will be protonated at an acidic (low) pH:
amino group has a basic pKa and will be protonated until basic pH (high) is achieved
carboxylic acid has an acidic pKa
COOH = COO− + H+
slightly more acidic than an carboxylic acids
The amino group has a basic pKa
NH4+ = NH3 + H+
Slightly less basic than in amines
Ionization of Amino Acids
low pH
the amino acid exists in a positively charged form (cation).
Ionization of Amino Acids
high pH
the amino acid exists in a negatively charged form (anion).
zwitterion
Between the pKa for each group, the amino acid exists in a zwitterion form, in which a single molecule has both a positive and negative charge.
Isoelectric Point (equivalence point, pI)
PI = (pK1 + pK2) / 2
In Zwitterions
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
As buffers prevent change in pH close to the pKa, glycine can act as a buffer in two pH ranges.
Ionizable Side Chains Also Have pKa and Act as Buffers
Ionizable side chains influence the pI of the amino acid.
Ionizable side chains can be also titrated.
Titration curves are now more complex, as each pKa has a buffering zone of 2 pH units.
How to Calculate the pI When the Side Chain Is Ionizable
Identify species that carries a net zero charge.
Identify the pKa value that defines the acid strength of this zwitterion: (pKR).
Identify the pKa value that defines the base strength of this zwitterion: (pK2).
Take the average of these two pKa values.
Amino Acids Polymerize to Form Peptides
Peptides are small condensation products of amino acids.
They are “small” compared with proteins (Mw < 10 kDa).
Naming Peptides: Start at the N-terminal
Numbering (and naming) starts from the amino terminus (N-terminal).
Using full amino acid names
Using the three-letter code abbreviation
For longer peptides (like proteins) the one- letter code
Peptides: A Variety of Functions
Hormones and pheromones
Neuropeptides
Antibiotics
Protection, e.g., toxins
Proteins are comprised of:
Polypeptides (covalently linked a-amino acids) + possibly:
- cofactors
- coenzymes
- prosthetic groups
- other posttranslational mods
cofactors
functional non-amino acid component
metal ions or organic molecules
coenzymes
organic cofactors
prosthetic groups
covalently attached cofactors
A Mixture of Proteins Can Be Separated
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
allows separation of a mixture of proteins over a solid phase (porous matrix) using a liquid phase to mobilize the proteins.
Proteins with a lower affinity for the solid phase will wash off first; proteins with higher affinity will retain on the column longer and wash off later.
Separation by Charge: Ion Exchange
proteins move through columns at rates determined by their net charge at the pH being used.
With cation exchangers, proteins with more negative net charge move faster
Separation by Size: Size Exclusion
Larger molecules pass more freely and appearing in the earlier fractions first using crosslinked polymer
Separation by Binding: Affinity
Protein mixture added to column contained polymer bound ligand for specific protein of interest.
Unwanted proteins are washed through the column and then protein of interested eluted by ligand solution
Electrophoresis for Protein Analysis
The electric field pulls proteins according to their charge.
The gel matrix hinders mobility of proteins according to their size and shape.
The gel is commonly polyacrylamide, so separation of proteins via electrophoresis is often called polyacrylamide gel electrophoresis, or PAGE
SDS PAGE Separates Proteins by Molecular Weight
SDS – sodium dodecyl sulfate – a detergent
SDS micelles bind to proteins and facilitate unfolding.
SDS gives all proteins a uniformly negative charge.
The native shape of proteins does not matter.
The rate of movement will only depend on size: small proteins will move faster.
Isoelectric Focusing Can Be Used to Determine the pI of a Protein
Protein sample may be applied to one end of a gel strip with an immobilized pH gradient.
After staining, proteins are shown to be distributed along pH gradient according to their PI values
Aromatic Amino Acids Absorb Light in a Concentration-Dependent Manner
Proteins typically have UV absorbance maxima around 275–280 nm.
Tryptophan and tyrosine are the strongest chromophores.
For proteins and peptides with known extinction coefficients (or sequences), concentration can be determined by UV-visible spectrophotometry using the Lambert-Beer law: A = ECL
Protein Sequencing
Edman degradation (classical method):
–successive rounds of N-terminal modification, cleavage, and identification
–can be used to identify protein with known sequence
Mass spectrometry (modern method):
–MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
–can be used to determine posttranslational modifications
Protein Sequences as Clues to Evolutionary Relationships
Differences indicate evolutionary divergences.
Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth.
native fold
a large number of favorable interactions within the protein
adopt a specific three-dimensional conformation with biological function
Hydrophobic effect
The 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 a-helices and b-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, especially those buried in the hydrophobic environment, strongly stabilize the protein.
Primary Structure
Peptide bond is a resonance hybrid of two canonical structures:
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 trans configuration
Secondary Structures
local spatial arrangement of the polypeptide backbone
a-helix
stabilized by hydrogen bonds between nearby residues of an n and n + 4 amino acids
B-sheet
stabilized by hydrogen bonds between adjacent segments that may not be nearby
random coil
Irregular arrangement of the polypeptide chain
Small hydrophobic residues such as Ala and Leu
strong helix formers
Pro
a helix breaker because the rotation around the N-Ca (φ-angle) bond is impossible
Gly
a helix breaker because the tiny R group supports other conformations
Helix Dipole in Peptide Bond
C−O (carbonyl) negative
N−H (amide) positive
helix has a large macroscopic dipole moment
B-sheets
planarity of the peptide bond and tetrahedral geometry of the a-carbon create a pleated sheet-like structure.
Sheet-like arrangement of the backbone is held together by hydrogen bonds between the backbone amides in different strands.
B-strands
Multiple B-sheets
Sheets are held together by the hydrogen bonding of amide and carbonyl groups of the peptide bond from opposite strands
Parallel and anti-parallel
B-turns
occur frequently whenever strands in B-sheets change the direction
180 degree turn is accomplished over four AA
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 B-turns.
Proline Isomers
Most peptide bonds not involving proline are in the trans configuration (>99.95%).
For peptide bonds involving proline, about 6% are in the cis configuration. Most of this 6% involve B-turns.
Catalyzed by proline isomerases
Circular Dichroism (CD) Analysis
signals from peptide bonds depend on the chain conformation and light or right light
