Lecture 8: Predicting/Testing Membrane Protein Structure/Topology

Question 1

What are amino acids joined by?

Answer 1

Polar Peptide bonds formed in a condensation reaction

Question 2

What is the exceptional AA?

Answer 2

Proline is an imino acid

Question 3

What are the amino acids in the alpha helical parts?

Answer 3

Non-polar/hydrophobic

Question 4

What is not possible in the TM regions?

Answer 4

Hydrogen bonding to water as there is none

Question 5

What neutralises the charge?

Answer 5

H bonds between the peptide bonds

Question 6

What is the pattern?

Answer 6

Regular H bonds between CO delta minus and NH delta plus. Between 1-4, 2-5, 3-6 etc forms alpha helix

Question 7

What do the R groups do?

Answer 7

Project out from the helix and are not involved in H bonding

Question 8

How many residues are there per turn at what length, what length per residue?

Answer 8

3.6, 5.4 A and 1.5 A

Question 9

How many AA are needed to span the bilayer in helical conformation?

Answer 9

20

Question 10

What is the length of the hydrophobic core of the bilayer?

Answer 10

30A

Question 11

Describe the basic structure of B-sheet membrane proteins

Answer 11

Every other AA R group projects in opposite orientation. Regular H bonding patterns between different strands. Each peptide bond is H bonded to another peptide bond in a different sheet. All the hydrophobic AAs could face outwards and all the hydrophilic ones could face inwards.

Question 12

How is an antiparallel B sheet formed?

Answer 12

with adjacent B strands running in opposite directions

Question 13

What is the smallest B barrel?

Answer 13

OmpX

Question 14

How many AA are needed to span the membrane with a B barrel and why is this different to alpha helix?

Answer 14

8-9 because in a more extended conformation

Question 15

What is alternated?

Answer 15

Whether they are lipid exposed or internal

Question 16

What are the inside facing lipids?

Answer 16

Hydrophilic and polar

Question 17

What does OmpX act as and how?

Answer 17

An adhesion protein as the protruding loops provide binding sites for other proteins with B strands

Question 18

What are the different hydrophobicity scales?

Answer 18

Kyte, Doolittle and Engelmann and Steitz

Question 19

How can you predict whether a membrane protein is likely to be integral?

Answer 19

By assigning each AA a hydrophobicity value and averaging over 15-20 AA.

Question 20

What does the hydrophobicity analysis allow?

Answer 20

Prediction of the number of TM spanning segments

Question 21

What is plotted?

Answer 21

The averages after moving the window along by one AA at a time

Question 22

Why can this method be difficult?

Answer 22

The cut off for a hydrophobicity TM domain can be arbitrary eg -1

Question 23

What does hydrophobicity analysis not work with and why?

Answer 23

B barrels as the hydrophobic/hydrophilic regions are alternate so are cancelled out

Question 24

Why is a smaller window not used?

Answer 24

Gets a very messy plot as the averages are more greatly affected by shifting the window by 1.

Question 25

Why aren’t all TM helices hydrophobic and give an example?

Answer 25

Not all hydrophobic stretches are part of the TM domains eg. Lac Y where some hydrophilic domains are part of the helix for protein-lipid interactions rather than protein-protein interactions.

Question 26

What two ways can the predictions be improved?

Answer 26

The positive inside rule and sequence alignments

Question 27

What is the positive inside rule?

Answer 27

Cytoplasmic loops of membrane proteins contain lots of positively charged AAs compared to extracytoplasmic loops. The positive regions are usually positioned a certain distance fro, the TM region so longer loops may not be positive far away

Question 28

What does the TOPPRED programme do?

Answer 28

Takes into account hydrophobicity and the positive inside rule to predict the number and orientation/topology of the protein.

Question 29

What can be positionally conserved?

Answer 29

AAs

Question 30

Which AAs are frequent in TM regions?

Answer 30

Leucine and isoleucine

Question 31

What different contacts can alpha helices make?

Answer 31

Protein-protein or protein-lipid

Question 32

What AA sequences are common for knobs into hole interaction?

Answer 32

GXXXG, GXXXA promotes interactions between helices

Question 33

What is the pattern of these sequences?

Answer 33

The GG and GA parts will be on the same face of the helix.

Question 34

In bovine cytochrome C oxidase, what is positioned along the helix so they can interact with each other via H bonds? What is the pattern?

Answer 34

Serines. They are positioned 2 turns of the helix from each other.

Question 35

Conserved H-bonds between polar residues in TM segments are important for what?

Answer 35

helix alignments