Session 2.4b - Group Work - Lecture 2 Flashcards
27th October 2017 11:00
Membrane Proteins
10) Apart from small changes of protein conformation, only two modes of mobility are permitted for integral membrane proteins. What are they?
- Rotational
2. Lateral
11) Proteins differ from phospholipids in not having a third mode of mobility
What is this mode and why do you think proteins do not exhibit this mode?
Proteins cannot FLIP FLOP, unlike phospholipids.
This is because proteins have a large hydrophilic structure which would need to move across the membrane, thus the thermodynamic energy is too great to permit that movement
12) Compared with the lipid constituents of the plasma membrane, why is the movement of protein in lipid bilayers more restricted?
Proteins have many more restrictions:
- they may have membrane protein associations, such as with each other (aggregates), which restricts their movement
- they may have associations with extra-membranous proteins (tethering), e.g. to the cytoskeleton, which restricts their motion by anchoring it
- they may have protein-protein interactions between neighbouring cells, which further anchors the protein
It also has
- lipid-mediated effects; so these proteins tend to be found in the fluid phase/cholesterol-poor environments where there is more fluidity, so they cannot move as freely as phospholipids.
13) a) Sketch a simple diagram of the Lipid Mosaic Model of the Singer Nicholson Model. Show proteins defined as peripheral and integral. Indicate their relative charge distributions relative to the internal part of the plasma membrane and the outward and inward facing surfaces label appropriately.
See Slide 11 Lecture 2
Extracytoplasmic Surface
Cytoplasmic surface
Hydrophilic - + - + - + -
Hydrophobic - no charge
Peripheral proteins on the surface or halfway through: hydrophilic parts outside, hydrophobic parts inside
Integral proteins all the way through: hydrophilic parts outside, hydrophobic parts inside
13) b) Briefly list the differentiating characteristics of peripheral and integral proteins.
Differentiating characteristics of Peripheral and Integral proteins.
• Peripheral
– Bound to surface
– Electrostatic and hydrogen bond interactions
– Removed by changes in pH or in ionic strength
• Integral
– Interact extensively with hydrophobic domains of the lipid bilayer
– Cannot be removed by manipulation of pH and ionic strength
– Are removed by agents that compete for non-polar interactions
e.g. detergents and organic solvents
13) c) Thinking about Session 01 on Homeostasis last week if there was significant loss in control of pH and increased ion concentration, briefly describe how might this affect the conformation of membrane proteins ?
Effects of change in pH and ionic concentration on protein conformation
Change in pH and ionic strength can remove peripheral proteins, but not integral proteins. This means the conformation of peripheral proteins can be changed but
14) In a plasma membrane, a phospholipid molecule may diffuse 200 um sec-1 laterally on average. This distance can be just about seen with the naked eye. In comparison, the average distances moved in one second by two transmembrane proteins, rhodopsin and fibronectin receptor, are 130 um and just 2.0 um, respectively.
a) What might account for the different mobilities of these two protein molecules?
* NB We will not ask assessment questions liked this. It is for encouraging thinking in Groupwork ! *
Rhodopsin is a very mobile protein.
Fibronectin is a peripheral glycoprotein that interacts with the extracellular matrix.
Fibronectin has a very low mobility because it is anchored to actin filaments on the inside of the plasma membrane through integrin, a transmembrane protein that links the extracellular matrix to the cytoskeleton.
So rhodopsin is able to move fast because it is a small protein not anchored to anything, whereas fibronectin moves slowly because it is anchored to something in the extracellular matrix, restricting its movement.
14) a) In a plasma membrane, a phospholipid molecule may diffuse 200 um sec-1 laterally on average. This distance can be just about seen with the naked eye. In comparison, the average distances moved in one second by two transmembrane proteins, rhodopsin and fibronectin receptor, are 130 um and just 2.0 um, respectively.
b) Why do you think the mobility of these two proteins is different?
* NB We will not ask assessment questions liked this. It is for encouraging thinking in Groupwork ! *
The rapid movement of rhodopsin is essential for fast signaling.
Fibronectin is involved in structural elements of the protein, thus, it is not required for it to move, in fact, you want it to be stationary.
Therefore, the mobility of these two proteins are different because they are linked to their function.
A Challenging Question: The PDGF receptor
15) NB We will not ask assessment questions liked this. It is for encouraging thinking in Groupwork !
The PDGF receptor (PDGF-R) is a plasma membrane protein that binds the growth factor, platelet-derived growth factor. This protein is identified in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as a polypeptide with an apparent molecular size of 180 kDa. Membrane preparations of cells were prepared as outlined in the table and were subjected to the following labelling protocols followed by SDS-PAGE to separate the cell proteins:
a) Lactoperoxidase/
125Iodine - a non-penetrating protein labelling reagent that labels proteins on accessible tyrosine residues with radioactive 125iodine.
b) Galactose oxidase - a non-penetrating carbohydrate labelling reagent. Transfers radioactive 3H onto galactose residues.
Labelling of a 180 kDa polypeptide was assessed.
In addition, the following qualitative assays were performed in each membrane preparation:
c) specific binding of 125I-labelled platelet-derived growth factor peptide(125I-PDGF)
d) release of a peptide containing the N-terminal amino acid sequence of the receptor on treatment with trypsin.
e) binding of a rhodamine-labelled antibody (fluorescent) which recognises a short amino acid sequence in the C-terminal of the PDGF-R protein.
The following results were obtained
See additional file: ICPP 2017 Workgroup Session 02 - Paramagnetic Spin Resonance PL PC - NO - Image
a) What can be concluded concerning the orientation of the PDGF-R protein in the plasma membrane? Draw a model of the proposed membrane topology of the PDGF-R.
No data?
15) NB We will not ask assessment questions liked this. It is for encouraging thinking in Groupwork !
The PDGF receptor (PDGF-R) is a plasma membrane protein that binds the growth factor, platelet-derived growth factor. This protein is identified in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as a polypeptide with an apparent molecular size of 180 kDa. Membrane preparations of cells were prepared as outlined in the table and were subjected to the following labelling protocols followed by SDS-PAGE to separate the cell proteins:
a) Lactoperoxidase/
125Iodine - a non-penetrating protein labelling reagent that labels proteins on accessible tyrosine residues with radioactive 125iodine.
b) Galactose oxidase - a non-penetrating carbohydrate labelling reagent. Transfers radioactive 3H onto galactose residues.
Labelling of a 180 kDa polypeptide was assessed.
In addition, the following qualitative assays were performed in each membrane preparation:
c) specific binding of 125I-labelled platelet-derived growth factor peptide(125I-PDGF)
d) release of a peptide containing the N-terminal amino acid sequence of the receptor on treatment with trypsin.
e) binding of a rhodamine-labelled antibody (fluorescent) which recognises a short amino acid sequence in the C-terminal of the PDGF-R protein.
The following results were obtained
See additional file: ICPP 2017 Workgroup Session 02 - Paramagnetic Spin Resonance PL PC - NO - Image
b) What further information would be required to confirm the topology of this protein in the plasma membrane and the site of adrenaline binding?
No data?
uestion: The PDGF receptor
15) NB We will not ask assessment questions liked this. It is for encouraging thinking in Groupwork !
The PDGF receptor (PDGF-R) is a plasma membrane protein that binds the growth factor, platelet-derived growth factor. This protein is identified in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as a polypeptide with an apparent molecular size of 180 kDa. Membrane preparations of cells were prepared as outlined in the table and were subjected to the following labelling protocols followed by SDS-PAGE to separate the cell proteins:
a) Lactoperoxidase/
125Iodine - a non-penetrating protein labelling reagent that labels proteins on accessible tyrosine residues with radioactive 125iodine.
b) Galactose oxidase - a non-penetrating carbohydrate labelling reagent. Transfers radioactive 3H onto galactose residues.
Labelling of a 180 kDa polypeptide was assessed.
In addition, the following qualitative assays were performed in each membrane preparation:
c) specific binding of 125I-labelled platelet-derived growth factor peptide(125I-PDGF)
d) release of a peptide containing the N-terminal amino acid sequence of the receptor on treatment with trypsin.
e) binding of a rhodamine-labelled antibody (fluorescent) which recognises a short amino acid sequence in the C-terminal of the PDGF-R protein.
The following results were obtained
See additional file: ICPP 2017 Workgroup Session 02 - Paramagnetic Spin Resonance PL PC - NO - Image
c) Why is the asymmetrical orientation of the PDGF-R important?
(No data?)
Different functions?
