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 K m –Meaning –Usage –Experimental determination –Manipulation?

V max –Meaning –Usage –Experimental determination –Manipulation? Kinetic parameters

k cat (turnover number) k cat /K m (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