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how can we test for the mobility of membrane proteins?
put antibodies on beads which will attach to protein of interest
how can we test for the mobility of protein?
laser trap, immobile proteins will not be displaced b/c of force
what is the movement of protein dependent on?
how do we know that membrane proteins are mobile and can be fused?
using rhodamine (mouse) and fluorescein (human) labeled membrane protein
what is used to measure lateral diffusion rates in membranes?
FRAP (flourescence recovery after photobleaching)
order the permeability of molecules
small hydrophobic, small uncharged polar, larger uncharged polar, ions
what does it mean that carriers have enzymatic qualities?
carrier proteins bind specific solutes, resulting in conformational changes
what is electrochemical gradient?
- sum of electrical potential difference and solute concentration gradient
- sometimes used in transport
what are the cellular processes driven by energy stored in ion gradients?
- chemiosmotic (uptake/efflux of nutrients, metabolites, salts)
- osmotic volume regulation (H2O follows ions)
- chemical (H+Na+ driven ATP synthesis
- cell homeostasis (pH regulation, efflux or sequestration of toxic solutes
- signal transduction (Ca entry, action potentials)
- mechanical (H+ driven flagellar rotation)
what are the characteristics of transporters and active transport?
- like enzymes without modifying substrate--binds to itself (kinase use ATP and put phosphate on substrate)
- conformational change is essential
- three ways to transport (ATP driven, light driven, coupled transporters)
what does the bacteriorhodopsin use as energy, substrate and distribution?
use light, H+ is the substrate, distributed in halobacteria
what does the photoredox use?
- light as energy
- H+ substrate
- photosynethic organisms
what does the electron transport chain NADH oxidase use?
H+, use redox potential, H+ substrate and distributed in mito and bacteria
ion trasnporting decarboxylases use?
use decarboxylation, Na is the substrate found in bacteria
H+ pyrophosphatases used how where ?
energy comes from pyrophosphate, H* in plants, fungi and bacteria
ATPases use what?
universal distribution, various ions and solutes, uses ATP
how is energy conserved in pumps?
in the form of transmembrane electrical or chemical gradient of the transported ion or solute. PE of these ion gradients drive energy requiring processes
what is another name for pumps b/c of its ability to transduce electromagnetic or chemical energy directly into transmembrane concentration gradients?
primary active transporters?
how does secondary transporters play a role in pumps?
use ion concentration created by pumps to drive other reactions
what does bacteriorhodopsin do?
it is a 7 pass transmembrane proton which change in retinal shape. use light to pump protons out of cell which creates proton gradient for ATP synthase to make ATP
describe the process of bacteriorhodopsin
- retinal in middle covalently attached to multipass protein
- when light passes through, retinal changes shape from trans to cis (isomerization), causes conformational change in protein
- Asp85 has low pKa (serves as proton acceptor) Asp96 provides proton (proton donor)
what are the three types of transport ATPases?
- F-type (V-type)
- F and P type generate electrical or chemical gradients across membranes
F-type distribution, substrate and function
- ATP synthesis or ATP driven H+ pumping
- in bacteria, chloroplast, inner mitochondria membrane, H+ substrate
- F1 is water soluble catalyzes ATP hydrolysis or synthesis
- F0 is embedded in membrane/passively conducts protons across bilayer
- stalk connects F1 to F0 to couple proton translocation to ATPsynthesis
V-type distribution and functions
- ATP driven H+ pumping, H+ substrate, found in archaea, eukaryotic membranes, pumps protons into organelles
- lysosomes, maintain low pH in cell compartments
where does ABC work, function substrate?
- ABC in plasma membrane, various solutes, solute transport, pump small molecules instead of ions
- Cl transport
what is the role of P-type?
- cation pumping, Na K, Ca, in plasma membrane and ER
- related multi-pass transmembrane pumps, phosphorylate themselves during cycle
what is unique about ATP powered pumps?
- move ions against gradient
- transmembrane proteins with ATP binding site on cytosolic side
- coupling ATP hydrolysis and transport (one proton is absorbed with light and one proton is transported into cell)
- ATP powered pumps generate ionic gradiens across cell membrane (cytosolic K is high and Na is low; Cl is low and Ca is very low)
what are some examples of p type pumps?
- Ca in SR in muscle relaxation
- Na/K ATPase maintains intracellular Ka and K in cells
- H ATPases maintain acidity of lyosomes
how do we determine the rotation of the subunits of ATPase?
- attach bead on isolated domain of ATpase, in presence of ATP, bead will rotate
- rotation of the gamma subunit insdie the alpha/beta subunits
describe the process of the ATPase
- H+ energy is stored in the bond energy of ATP
- H+ move into rotor, protonate forms can attach and rotate motor exit (proton energy into mechanical rotational energy)
- rotational energy is transmitted to shaft attached to motor, goes into ATPase which catalyzes the formation of ATPase
- position of shaft influences the surrounding subunits (these changes drive synthesize of ATP from ADP)
what drives the chemioosmotic cycle?
proton gradient which drives rotation
what do P Type Na K ATPase uses to catalyze electrogenic antiport of K and Na?
ATP hydrolysis accounts for 30% of ATP hydrolysis in most cells (70% in brain)
describe the process of Na/K pump
3Na binds, ATP binds, hydrolyzes, phosphorylation, Na released 2 K binds, which triggers release of phosphate
what is the function of Na-K ATPase?
maintain gradient that encompases PE for work; ATP binding leads to release of K on cytoplasm and regeneration of E1 conformation
what is the calcium P-Type intracellular pump composed of?
10 transmembrane alpha helices that regulates osmolarity and membrane potential
what is the occluded state of Ca?
when Ca not accessible on either side of membrane to prevent protein leak