Ch.2 - Amino Acids, Peptides, and Proteins

All 20 common amino acids are ___ amino acids

α-amino acids — they all have a carboxyl group and amino group bonded to the same α-carbon, differing only in their R groups

The α-carbon of amino acids is a chiral center because

it is bonded to four different groups (carboxyl, amino, R group, and H); glycine is the exception with H as its R group

Stereoisomers of amino acids that are nonsuperposable mirror images

enantiomers; classified as L or D based on absolute configuration relative to L- or D-glyceraldehyde

Stereoisomer configuration in proteins

nearly all L stereoisomers; less than 1% D-amino acid residues, which are introduced post-synthesis by enzyme-catalyzed reactions

Why cells produce L amino acids specifically

enzyme active sites are asymmetric, making reactions stereospecific

Five classification groups of amino acids by R group polarity

nonpolar aliphatic, aromatic, polar uncharged, positively charged (basic), negatively charged (acidic)

Nonpolar aliphatic R group amino acids

Glycine, Alanine, Proline, Valine, Leucine, Isoleucine, Methionine — hydrophobic; stabilize protein interior via hydrophobic effect

Why proline is structurally distinctive

its side chain forms a cyclic structure with the α-amino group (secondary/imino group), restricting polypeptide flexibility

Aromatic R group amino acids and UV absorption

Phenylalanine, Tyrosine, Tryptophan; Trp and Tyr absorb strongly at ~280 nm, accounting for most protein UV absorbance

Polar uncharged R group amino acids

Serine, Threonine (hydroxyl), Cysteine (sulfhydryl), Asparagine, Glutamine (amide groups) — form H-bonds with water

Cystine formation and function

two Cys residues oxidized to form a disulfide bond (cystine); strongly hydrophobic and form covalent cross-links stabilizing protein structure

Positively charged (basic) R group amino acids at pH 7

Lysine (ε-amino), Arginine (guanidinium), Histidine (imidazole, pKa ~6.0); only His can be + or uncharged at pH 7

Negatively charged (acidic) R group amino acids at pH 7

Aspartate and Glutamate — each has a second carboxyl group in its R group

Uncommon amino acids created by postsynthetic modification examples

4-hydroxyproline (in collagen), γ-carboxyglutamate (in prothrombin), desmosine (in elastin from 4 Lys residues)

Selenocysteine and pyrrolysine differ from other uncommon amino acids because

they are incorporated during protein synthesis via specialized adaptation of the genetic code, not by postsynthetic modification

Zwitterion

dipolar ionic form of amino acids at neutral pH; has both + (protonated amino) and − (deprotonated carboxyl) charges simultaneously; net charge depends on pH

Isoelectric point (pI)

the pH at which an amino acid or protein has zero net charge; for amino acids without ionizable R groups

pKa of α-carboxyl group vs. typical carboxyl group

~2.0–2.4 in amino acids vs. ~4.8 for simple carboxylic acids; lower because the nearby protonated amino group withdraws electrons

How the Henderson-Hasselbalch equation applies to amino acid titrations

at the midpoint of each titration stage, pH = pKa of that group; the pKa equals the pH where that group is 50% ionized

Peptide bond

a substituted amide linkage formed by condensation (dehydration) between the α-carboxyl group of one amino acid and the α-amino group of another; releases H₂O

Average half-life of peptide bonds under intracellular conditions

~7 years; hydrolysis is thermodynamically favorable but kinetically very slow due to high activation energy

Convention for writing peptide sequences

amino-terminal (N-terminal) end on the left, carboxyl-terminal (C-terminal) end on the right; named starting from N-terminus

Polypeptides vs. proteins by molecular weight

polypeptides generally

Oligomeric protein and protomer

an oligomeric protein has ≥2 identical polypeptide units; the identical repeating unit (which may itself consist of multiple chains) is the protomer

Conjugated protein

contains non-amino acid chemical components (prosthetic groups) in addition to amino acids; examples include glycoproteins (carbohydrates), hemoproteins (heme), metalloproteins (metal ions)

Average molecular weight of an amino acid residue in a protein

~110 Da (average amino acid ~128 Da minus 18 Da for water lost per peptide bond)

Salting out

selective precipitation of proteins using high salt concentration (e.g., ammonium sulfate), which lowers protein solubility; used as an early fractionation step

Dialysis in protein purification

separates proteins from small solutes using a semipermeable membrane; retains large proteins while allowing small molecules to equilibrate with the external solution

Ion-exchange chromatography principle

separates proteins by net charge; cation exchangers bind positively charged proteins (retarding their migration), anion exchangers bind negatively charged proteins

Size-exclusion chromatography (gel filtration) principle

separates by size — larger proteins elute first because they cannot enter pores of the beads; smaller proteins take a longer, more labyrinthine path

Affinity chromatography principle

separates proteins by binding specificity to a ligand attached to beads; unwanted proteins wash through, and the protein of interest is eluted by free ligand or high salt

HPLC advantage over standard column chromatography

high-pressure pumps increase speed and use higher-quality materials, reducing diffusional spreading and greatly improving resolution

SDS-PAGE principle and what it measures

SDS binds ~1.4× protein weight (one molecule per residue), conferring uniform negative charge and partial unfolding; separates proteins almost exclusively by mass (molecular weight)

Isoelectric focusing

separates proteins by pI using a pH gradient gel; proteins migrate until they reach the pH matching their pI, where net charge = 0

Two-dimensional electrophoresis

combines isoelectric focusing (separates by pI) and SDS-PAGE (separates by molecular weight) sequentially; resolves thousands of proteins in a complex mixture

Specific activity

enzyme units per milligram of total protein; increases with each purification step and becomes maximal and constant when the protein is pure

1 unit of enzyme activity

the amount catalyzing transformation of 1.0 µmol substrate to product per minute at 25°C under optimal conditions

Lambert-Beer Law

A = εcl; where A = absorbance, ε = molar extinction coefficient (L/mol·cm), c = concentration (mol/L), l = path length (cm); absorbance is directly proportional to concentration

Proteins absorb UV light at 280 nm because

tryptophan and tyrosine (and minimally phenylalanine) absorb at this wavelength; widely used to estimate protein concentration

Four levels of protein structure

primary (amino acid sequence and covalent bonds including disulfide bonds); secondary (local recurring patterns); tertiary (overall 3D folding of one polypeptide); quaternary (arrangement of multiple subunits)

Primary structure determines

the 3D structure of a protein, which in turn determines its function; sequence variants can drastically alter or abolish activity

Mass spectrometry in proteomics

measures mass-to-charge ratio (m/z) of ionized molecules; MALDI MS ionizes proteins from a matrix with laser pulses; ESI MS disperses proteins from solution into charged droplets; both transfer macromolecules to gas phase for analysis

Tandem MS (MS/MS) for sequencing

protein is digested (often with trypsin), peptides are isolated in MS-1, fragmented in a collision cell, and fragments analyzed in MS-2; successive peaks differ by mass of one amino acid, revealing sequence

Proteome

the entire complement of proteins expressed in a cell, including relative abundance estimates; can be analyzed by LC-MS/MS in approximately one hour

Edman degradation

classical method for protein sequencing from N-terminus; uses chemical reagents (e.g., FDNB, dansyl chloride) to label and sequentially remove one residue at a time

Proteases used in protein sequencing

trypsin cleaves C-terminal to Lys or Arg; chymotrypsin cleaves C-terminal to Phe, Trp, Tyr; cyanogen bromide cleaves C-terminal to Met

Solid-phase peptide synthesis (Merrifield method)

peptide built C-terminus to N-terminus on an insoluble resin support, one amino acid at a time with protective groups (e.g., Fmoc); up to ~100 residues feasible

Homologous proteins (homologs)

proteins from related evolutionary origin sharing sequence similarity and often structural/functional features; paralogs are homologs within the same species; orthologs are homologs from different species

Signature sequence

a short amino acid insertion or motif found only in a specific taxonomic group; used to identify evolutionary relationships (e.g., 12-residue insertion in EF-1α/EF-Tu proteins shared by archaea and eukaryotes but absent in bacteria)

Consensus sequence

the most common amino acid (or nucleotide) at each position when multiple related sequences are aligned; conserved positions often represent functionally critical residues

Conservative substitution in sequence alignment

replacement of one amino acid by another with similar chemical properties (e.g., Asp → Glu); receives higher alignment score than nonconservative substitution