Nonenzymatic Protein Function & Analysis

Question 1

Three Types of Ion Channels

Answer 1

• All permit facilitated diffusion of charged particles.
• Ungated Channels: Have no gates and are therefore unregulated.
• Voltage-Gated Channels: Are regulated by the membrane potential change near the channel; closed under resting conditions, opened due to membrane depolarization.
• Ligand-Gated Channels: The binding of a specific substance or ligand to the channel causes it to open or close.

Question 2

Describe G Protein-Coupled Receptors

Answer 2

• Integral membrane proteins involved in signal transduction characterized by their seven membrane-spanning alpha helices.
• Uses a heterotrimeric G protein (alpha, beta, gamma subunits) to transmit signals to an effector in the cell.
• Gs stimulates adenylate cyclase, which converts ATP to cAMP, thereby increasing levels of cAMP in cell.
• Gi inhibits adenylate cyclase; decreases levels of cAMP in cell.
• Gq activates phospholipase C; ultimately produces IP3 which opens calcium channels in the ER and increases calcium levels in cell.
• In its inactive form, alpha subunit binds GDP in complex with beta and gamma subunits. When ligand binds to GPCR, GDP is replaced with GTP, and alpha subunit dissociates from beta and gamma subunits. Activated alpha subunit alters activity of adenylate cyclase or phospholipase C. Once GTP on activated alpha subunit is dephosphorylated to GDP, alpha subunit rebinds to the beta and gamma subunits, rendering G protein inactive.

Question 3

Electrophoresis (Protein Isolation)

Answer 3

• Subjects compounds to electric field, moving them according to net charge and size.
• Negatively charged compounds migrate towards positively charged anode; positively charge compounds migrate towards negatively charged cathode.
• Small, highly charged molecules placed in large electric field migrate faster through polyacrylamide gel; big/convoluted, electrically neutral molecules placed in small electric field migrate slower through medium.

Question 4

Native PAGE

Answer 4

• Method for analyzing proteins in their native states.
• Limited by the varying mass-to-charge and mass-to-size ratios of cellular proteins, as multiple different proteins may experience the same level of migration.
• Functional native protein can be recovered from gel after electrophoresis if gel has not been stained, as most stains denature proteins.
• Most useful to compare molecular size or charge of proteins known to be similar in size from other analytic methods like SDS-PAGE or size-exclusion chromatography.

Question 5

SDS-PAGE

Answer 5

• Method for separating proteins on the basis of relative molecular mass using sodium dodecyl sulfate (SDS: detergent that disrupts all noncovalent interactions).
• SDS treatment denatures proteins and creates large chains with net negative charges.

Question 6

Isoelectric Focusing

Answer 6

• Method for separating proteins on the basis of their isoelectric point using gel with pH gradient (acidic gel at positive anode, basic gel at negative cathode, neutral in the middle).
• When electric field is generated across gel, positively charged proteins migrate towards cathode, and  negatively charged proteins migrate towards anode.
• Protein takes on a neutral charge and stops moving where pH of gel equals protein’s isoelectric point (pH = pI).

Question 7

Chromatography (Protein Isolation)

Answer 7

• Techniques that require the homogenized protein mixture to be fractionated through a porous matrix.
• Preferred over electrophoresis when large amounts of protein are being separated.
• Works under the principle that a compound with a high affinity for the stationary phase will barely migrate at all (high retention time), while a compound with a high affinity for the mobile phase will migrate much more quickly (low retention time).
• Sample placed on stationary phase or adsorbent (solid medium), mobile phase is run through stationary phase (allowing sample to elute through stationary phase).

Question 8

Column Chromatography

Answer 8

• Column is filled with polar silica or alumina beads as an absorbent, and gravity moves nonpolar solvent down the column.
• The less polar the compound, the faster it can elute through the column (low retention time); polar compounds elute much slower, if at all.

Question 9

Ion-Exchange Chromatography

Answer 9

• Beads in the column are coated with charged substances to attract or bind compounds with the opposite charge.
• A positively charged column will attract and hold a negatively charged protein as it passes through the column, increasing its retention time or retaining it completely.
• After collection, a salt gradient can be used to elute the retained protein.
• Anion-Exchange Chromatography uses positively charged beads.
• Cation-Exchange Chromatography uses negatively charged beads.

Question 10

Size-Exclusion Chromatography

Answer 10

• Beads in the column contain tiny pores of varying sizes, which can hold smaller compounds, increasing their retention times; larger compounds will elute faster.

Question 11

Affinity Chromatography

Answer 11

• Beads are coated with a target (receptor, antibody, substrate, etc.) that binds the protein of interest and retains it in the column.
• Retained protein can be eluted by washing column with free targets; note that recovered substance may be bound to the eluent.

Question 12

Southern, Northern, Western Blotting

Answer 12

• SNoW DRoP.

• Southern Blot: DNA.
• Northern Blot: RNA.
• Western Blot: Protein.