Biochemistry Exam 2

Protein structures, amino acid conformation and Carbohydrates

Peptide bond conformation

trans

monomeric protein

one polypeptide chain

homomultimer

one kind of chain

heteromultimer

two or more kind of chains

primary structure

amino acid sequence

secondary structure

local structures stabilized by H bonds (alpha helix and beta strands)

tertiary structure

overall 3D shape of folded structure (backbone, side chains)

quatenary structure

subunit organization (domains)

proteins are least soluble @

isoelectric point

Dialysis

protein purification method separating proteins based on diffusible solutes ability to pass semi permeable bag membrane

Ion exchange chromatography

protein purification method that separate amino acids on cation or anion exchange column based on charge

Size Exclusion chromatography/ gel filtration

protein purification method that allow larger molecules to escape from gel beads in a column than smaller separating protein by size

Electrophoresis (SDS Page)

Protein purification method that applies SDS detergent to protein that causes them to break into negatively charged short peptide chains and drawn towards anode (positive charge) separated based on molecular mass

2D Gel electrophoresis

Protein purification method that separates macromolecules based on isoelectric points and placed in SDS Page slab and into SDS gel where they’re separated by size

Affinity chromatography

Protein purification method that separates proteins based on placing target ligands into a column and allow proteins to pass through binding to corresponding ligand - ligands are then freed via concentrations of free ligand

Hydrophobic Interaction Chromatography (HIC)

protein purification method that exploits hydrophobic natures of proteins

High Performance Liquid Chromatography (HPLC)

protein purification method that uses nonpolar liquid as stationary phase

Ultracentrifugation

protein purification method that used high density gradient to separate proteins

Protein sequencing step 1

separation of chains via extreme pH

Protein sequencing step 2

cleavage of disulfide bridges via sulfhydryl reducing agents (Mercaptoethanol)

Protein sequencing step 3

Identify N and C termini residues via edman reagents and carboxypeptidase respectively

Protein sequencing step 4 and 5

fragmentation and and analysis of polypeptide chains (trypsin)

Protein sequencing step 6

reconstruct sequence (Edman degradation)

Electrospray Ionization (ESI - MS)

protein sequencing method that sprays macromolecules with positively charged fine droplets charging the molecules that can be translated to M/Z values signifying number of charges per molecule

Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS)

protein sequencing method that uses laser to excite molecules to micro explosions and ejects them into gas phase containing positively charged species that can be selected by MS for mass analysis

Electrospray Ionization (ESI - MS) Limitations

Can be too complex to read due to multiple charge states

Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS) Limitations

limited to single charged ions and less suitable for direct sequencing

Homologous proteins

proteins sharing similar degree of sequence and structural resemblance or perform same function in different organisms

Orthologous proteins

proteins in different species that have homologous sequence (and similar function) - cytochrome C

Paralogous proteins

proteins found within a single species that have homologous amino acid sequences (ie alpha and beta hemoglobin)

Prosthetic group

non amino acid part of protein necessary for its function