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)
Stereoisomers of -amino acids All amino acids in proteins are L-amino acids, except for glycine, which is achiral.
Alternative Representation of Amino Acids All L-amino acids in proteins are S, except for cysteine, which is R. Leucine Cysteine
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 (g-Glu-Cys-Gly) makes the inside of the cell a highly reducing environment.
Hydroxyl-Containing Amino Acid Side Chains Serine Threonine Tyrosine
Tyrosine, Serine and Threonine Can Be Phosphorylated in Proteins Example: Tyrosine
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
cis and trans Isomers The trans isomer is generally more stable because of steric crowding of side chains in the cis isomer.
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/1a Myoglobin
Initiating Amino Acid in Translation N-Formylmethionine in prokaryotes Just methionine in eukaryotes
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.
Polyampholyte Character of a Tetrapeptide and Isoelectric Points Group pKa a-NH3+ 9.7 Glu g-COOH 4.2 Lys e-NH3+ 10.0 a-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).
Electrophoresis through polyacrylamide gel in which there is a pH gradient. Isoelectric Focusing
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 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