Membrane dynamics

• Degree of fluidity is similar in all membranes– Cells regulate composition based on growth

conditions

Membrane dynamics• Experiment on lateral diffusion

– Attach fluorescent probes to head group to monitor

– Tag all lipids on outer membrane– Bleach the probe– Measure fluorescence return

– FRAP: fluorescence recovery after photobleaching

Areas of lower dynamics?

• Structural proteins within the cell ‘anchor’ certain proteins in place

• Lower degree of lateral diffusion

Areas of lower dynamics?

• ‘Rafts’– Thick, more ordered

microdomains– Exclude

glycerophospholipids (shorter sat/unsat chains)

• Sphingolipids (long FA chains…mostly saturated)

• Cholesterol• Integral membrane

proteins

Lipid rafts

• Typically detergent-insoluble

• Serves to sequester a subset of proteins together…other reasons?

Extreme case of membrane segregation

• Caveolae: physical distortion of the membrane

• Segregates transmembrane signaling molecules, etc.

• eg. insulin receptor

Exam review

• Questions?

Kinetic parameters

• Km

– Meaning– Usage– Experimental determination– Manipulation?

• Vmax

– Meaning– Usage– Experimental determination– Manipulation?

Kinetic parameters

• kcat (turnover number)

• kcat/Km (specificity constant)

• Inhibition constants– Competitive– Non-competitive– Irreversible?

Kinetic parameters

General aspects of catalysis

• How do catalysts affect kinetics?

• Mechanisms:– Give two (more?) ways a positively-charged

residue in an active site might influence a reaction