Lecutre - Protein Shit

Question 1

Concentration of oxygen is saline vs heamogolin in blood

Answer 1

The concentration of oxygen in saline solution is limited to ~ 0.2 mmol/L
- while the concentration of haemoglobin in blood is ~ 5 mmol/L.

Question 2

Highly active tissue, e.g. exercising muscle or the brain, is limited by…

Answer 2

… availability of oxygen

Question 3

There is strong evolutionary pressure for

Answer 3

Efficient oxygen transport and delivery

Question 4

Active tissues use more ____ then _____ can deliver

Answer 4

Active tissues can use more O2 then blood can deliver

Question 5

Myglobin meets challenge of needing lots of oxygen by…

Answer 5

… storing oxygen in muscle against Burts of high requirement

Question 6

Globin

Answer 6

• myomolgobin is red and stored in tissues

Question 7

How much myoglobin in blood

Answer 7

Human muscles have 0.5-0.7 mmol/L myoglobin, enough for about 7 seconds of intense activity. After this store is exhausted the tissue depends on oxygen delivery from the lungs.

Question 8

Primary protein structure of myoglobin

Answer 8

150 amino acids

Question 9

Protein secondary structure of myoglobin

Answer 9

Eight a-helices A-H and connecting loops (AB, BC, etc.)

Question 10

Tertiary structure of myoglobin

Answer 10

globin fold with a hydrophobic pocket

Question 11

Myoglobin quanternary structure

Answer 11

monomeric (a single polypeptide chain)

Question 12

What does the ancient globin fold provide?

Answer 12

• hydrophobic pocked (see Val E11 and Phe CD1, next slide) to bind a haem group.

Question 13

Haem is a…

Answer 13

…prosthetic group or cofactor

Question 14

Structure if Haem

Answer 14

• includes pyrrole rings linked together in a plane
• iron has 6 coordinate bonds - four to nitrogen atoms of the Haem, one to a nitrogen atom of histidine F8 the globin, one to O2
• binding of oxygen to Fe2+ is a reverable reaction

Question 15

What gives Haem its red colour

Answer 15

• the molecular electronic orbitals of Haem give it a red colour

Question 16

Binding of oxygen to the Fe2+ is a….

Answer 16

.. reversible intereaction

Question 17

Haem

Answer 17

Question 18

Spectroscopy quantifies..

Answer 18

..dissolved molecules

Question 19

Haem is rich in..

Answer 19

Carbon and nitrogen
- they all bind an iron atom where oxygen binds

Question 20

Haem e planar?

Answer 20

Nah not quite - close to flat with fe in middle
HisF8 holds it in place - can also sense what’s going on thriugh bond
- side chains prevent oxygen from binding straight up and binding too strongly in an ideal postition

Question 21

Which interactions break and reform dousing myoglobins function

Answer 21

• His E7’s interaction with oxygen
• oxygens interation with iron

Question 22

What measures oxygens binding to myoglobin

Answer 22

Spectroscopy

Question 23

How spectroscopy quantifies dissolved molecules - yappage

Answer 23

• higher concentration = less transmitted light = higher absorbance
• beer-lambert law converts from absorbance to concentration
• monochromator - breaks light into its diff coulours - pushes known amount of light into solution - certain amout gets absorbed
• when converting between nitrogen bound and oxygen bound - can say the haemoglobin isn’t changing - only whether its in an oxygen bound or oxygen free state

Question 24

What measures binding and unbinding of Haem ogle in

Answer 24

Spectroscopy

Question 25

Spectroscopy of globin measure oxygen binding

Answer 25

- shape of spectrum differs with colour and with chemical nature of solute - protein is colourless (but has UV absorbance) - Haem has visible absorbance (and therefore colour) that differs between bright red oxyhaemoglobin (HbO2)dull red deoxyhaemoglobin (Hb)

Question 26

Haem Fe2+ is attached to globin protein by

Answer 26

Co-ordinate linkage to His F8

Question 27

His E7 is located where and why

Answer 27

..on the opposite side of haem and distorts binding of gas molecules to 6th co-ordination position of haem Fe2+ - this reduces the binding affinity of oxygen to myoglobin, making it easier to release oxygen to the muscle cell

Question 28

Myoglobin and haemoglobin both show ______ ______ of oxygen affinity

Answer 28

Myoglobin and haemoglobin both show ‘allosteric control’ of oxygen affinity - allosteric means ‘without overlapping’ - allosteric builds on steric hindrance - the impossibility of two atoms occupying the same space

Question 29

What does lactate do the myoglobins affinity for oxygen?

Answer 29

Lactate decreases myoglobins affinity for oxygen but does not bind where oxygen bins - this shifts the curve right wards - lactate build up in muscles promotes oxygen release from myoglobin, increasing O2 availability for respiration

Question 30

Graph of myoglobin and haemoglobin differing in O2 binding

Answer 30

- myoglobin is saturated with O2 at low pO2 only releasing O2 to muscle cells when the cellular pO2 is very low - this is shown in a hyperbolic curve - this suits its function as a ‘back-up’ store of O2 in muscle cells Mb + O2 = MbO2

Question 31

The partial pressure (pO2) of oxygen in lungs vs resting muscle

Answer 31

100 Torr in lungs 20 Torr in resting muscle

Question 32

Hamaglobion oxygen binding curve shape

Answer 32

Sigmoidal

Question 33

Compared to myoglobin, hemoglobin funcitoning as an O2 transporter in blood evolved a much weaker….

Answer 33

… binding affinity for oxygen

Question 34

The availability of O2 to cellular proteins depends on:

Answer 34

- the pO2 in the local environment - the binding affinity of O2 to haemoglobin

Question 35

How haemoglobins function means it has envelopes to bind O2 less tightly then myoglobin

Answer 35

- bind O2 in the vicinity of the lungs where the pO2 is 100Torr - release the O2 in the vicinity of peripheral tissues where the pO2 is 20 Torr - it can also change shape, ‘duping’ oxygen in peripheral tissues

Question 36

Which binds haemoglobin more tight?

Answer 36

Myoglobin

Question 37

Haemoglobin has evolved to be a (Structure)

Answer 37

Tetramer - four glob in proteins associate together non-covalently - each globin protein contains one haem and each can bind one O2 - 1,2,3,4 O2 can bind per one Hb tetramer

Question 38

Myoglobin (a monomer) acts as a

Answer 38

O2 store in muscle

Question 39

Structure difference of deoxyhaemoglobin and oxyhemoglobin

Answer 39

Question 40

When was the MWC concerted model proposed and who by?

Answer 40

Proposed in 1956 by Monod, Wyman and Changeux

Question 41

MWC, concerted model

Answer 41

- Subunits can be in a low-activity, tense (T) or high-activity, relaxed (R) conformation. - All subunits must be in the same state (either T or R). - Binding each successive substrate (S) shifts equilibrium in favour of R. - Inhibitors stabilise the T form. - Activators stabilise the R form.

Question 42

KNF, sqential model ([prolly not in exam)

Answer 42

• One substrate binding induces a T → R conformational change in only one subunit. • This conformational change influences the neighbouring subunits (i.e. cooperativity), changing their affinity for substrate. Many conformations are possible. • This explains both positive and negative cooperativity.

Question 43

Similarities between haemoglobin and myoglobin

Answer 43

• Oxygen binds to iron of haem. • Shift from dull to bright red allows monitoring O 2 binding. • Affinity for oxygen is altered by molecules (e.g. lactate to myoglobin) binding elsewhere (allosteric control).

Question 44

Myoglobin vs haemoglobin - differences

Answer 44

• Monomer versus tetramer • Tighter, hyperbolic versus weaker, sigmoidal binding curve • Store in tissue versus transport molecule

Question 45

Cooperatively

Answer 45

- requires multiple interacting subunits - gives a sigmoidal binding curve - shifts binding affinity (and the steep part of the binding curve) to a physiologically relavant oxygen concentration