Protein Quantitation Techniques & Homology Modeling PDF

Other protein quantitation techniques Colorimetric copper-based assays o Biuret Assay, Lowry Assay, & Bicinchoninic Acid (BCA) Assay Biuret Assay → Proteins with at least 3 amino acid residues form a complex with Cu2+ in an alkaline environment, consequently reducing Cu2+ to Cu1+ → The reaction prod...

Other protein quantitation techniques Colorimetric copper-based assays o Biuret Assay, Lowry Assay, & Bicinchoninic Acid (BCA) Assay Biuret Assay → Proteins with at least 3 amino acid residues form a complex with Cu2+ in an alkaline environment, consequently reducing Cu2+ to Cu1+ → The reaction produces a faint blue to purple color. The intensity of the color produced, measured at 540 nm, is directly proportional to protein concentration → Can detect higher protein concentrations than BCA Assay and Lowry Assay Other protein quantitation techniques Colorimetric copper-based assays BCA displaces proteins bound to Cu1+ BCA Assay → After the Biuret reaction, reduced copper (Cu1+ or cuprous ion) further reacts with bicinchoninic acid (BCA), resulting in an intense purple color measured at 562 nm (or any wavelength between 550 and 570 nm) → Can detect lower protein concentrations than the Biuret assay → Compatible with protein samples containing up to 5% surfactants/detergents Other protein quantitation techniques Colorimetric copper-based assays Biuret reaction Lowry Assay → After the Biuret reaction, a phosphomolybdic-phosphotungstic acid solution called Folin-phenol reagent is added and binds to the peptide-copper complex, producing an intense blue color, measured at 750 nm (or any wavelength between 650 nm and 750 nm) → Blue color is attributed to the heteropoly-molybdenum blue formed by the transfer of electrons from the peptide-copper complex to the phosphomolybdic-phosphotungstic acid complex → Can detect lower protein concentrations than the Biuret assay Homology Modeling of Protein Structure Homology modeling → Predicts the unknown three-dimensional structure of a protein by searching for another protein (Template) with known 3D structure whose sequence is highly similar to that of the unknown protein (Target or query) → Homologous or evolutionarily related proteins have similar sequences and similar 3D structure → Conservation of protein structure > conservation of amino acid sequence among homologues: even with only 30% sequence similarity, 2 proteins can still have similar 3D structure → Determining the structure of a protein is integral for understanding its function/s Homology modeling → Protein structure-function relationship is demonstrated in Sickle Cell Anemia Hemoglobin function is to bind oxygen. Mutant hemoglobin can still bind oxygen, but in low oxygen conditions, the mutant is prone to aggregation which causes deformation of red blood cells (where the hemoglobin protein is located) Levels of Protein Structure Levels of Protein Structure Amino acids Amino acids Tyr Primary structure → Peptide or Oligopeptide: Short chain of (2 to 20) amino acids → Polypeptide: Longer polymer with 50 amino acids or more, that can be in the folded state or not → Protein: Sometimes used interchangeably with polypeptide. Generally, however, this term refers to the folded, functional molecule composed of one polypeptide or multiple polypeptides Primary structure → Peptide bond is an amide bond formed between two amino acids through a dehydration synthesis reaction → N- to C-terminus synthesis: Proteins are always synthesized in a directional manner starting with the amine tail and ending with the carboxylic acid tail. New amino acids are always added onto the carboxylic acid tail, never onto the amine of the first amino acid in the chain. Primary structure → Peptide bond is an amide bond formed between two amino acids through a dehydration synthesis reaction → N- to C-terminus synthesis: Proteins are always synthesized in a directional manner starting with the amine tail and ending with the carboxylic acid tail. New amino acids are always added onto the carboxylic acid tail, never onto the amine of the first amino acid in the chain. Secondary structure → Local folded structures within a polypeptide formed by hydrogen bonding between atoms (Specifically, the C=0 group and the N-H group) of the peptide backbone → Side chains (R groups) are not involved in the formation of secondary structures Secondary structure R groups pointed away from the helix → Alpha-helix → Dotted lines show hydrogen bonds formed between C=O groups and N-H groups in the polypeptide backbone Secondary structure → Parallel and anti-parallel beta-pleated sheets Anti-parallel Parallel Secondary structure → Beta-pleated sheets Other less common secondary structures → Beta-turns, 310 helix, pi-helix → Beta-turn → Diameter: pi-helix > alpha-helix > 310 helix Tertiary structure → Overall 3D structure of the polypeptide → Tertiary structure is held together by different chemical interactions between 2 side chains or between a side chain and the peptide backbone atoms (with water molecules) (and negatively) Tertiary structure Tertiary structure Quaternary structure → The quaternary structure of a protein is formed by the assembly of multiple polypeptides, as in the case of antibody molecules (Shown below) o Homo-multimer/oligomer: a functional protein formed by 2 or more identical subunits or polypeptide chains o Hetero-multimer/oligomer: a functional protein formed by 2 or more different subunits or polypeptide chains → Hetero-multimeric protein Quaternary structure → The quaternary structure of a protein is formed by the assembly of multiple polypeptides o Homo-multimer/oligomer: a functional protein formed by 2 or more identical subunits or polypeptide chains o Hetero-multimer/oligomer: a functional protein formed by 2 or more different subunits or polypeptide chains → 4 different homo-multimeric proteins X-ray crystallography → Experimental technique considered as gold standard for determining 3D structure of proteins Protein crystal Ramachandran plot → Plot of the phi and psi angles of each amino acid of a protein o Phi angle (Φ): Angle between alpha-carbon and amino group o Psi angle (ψ): Angle between alpha-carbon and carboxyl group → Developed by Ramachandran, the plot shows the allowable phi and psi angles for amino acids so that the protein can form certain secondary structures PB Φ PB ψ Peptide bond (PB) Central or alpha carbon of this amino acid Ramachandran plot → Plot of the phi and psi angles of each amino acid of a protein o Phi angle (Φ): Angle between alpha-carbon and amino group o Psi angle (ψ): Angle between alpha-carbon and carboxyl group → Developed by Ramachandran, the plot shows the allowable phi and psi angles for amino acids so that the protein can form certain secondary structures → 1 dot = 1 amino acid in the chain Ramachandran plot → Plot of the phi and psi angles of each amino acid of a protein o Phi angle (Φ): Angle between alpha-carbon and amino group o Psi angle (ψ): Angle between alpha-carbon and carboxyl group → Developed by Ramachandran, the plot shows the allowable phi and psi angles for amino acids so that the protein can form certain secondary structures