nm0442: today we're going to s-, cover structural and functional relationships 
in proteins and i'm sure as most of you will really all be aware this is going 
to be er you know we're going to only cover a very small proportion of of er 
structural and functional relationships in proteins proteins are of course the 
sort of workhorses the machines of the cells and they perform all of the 
reactions that are compatible with life including those ones listed there er 
and of course there are very many relevant medical examples to how the 
structure and function of a protein is related to the normal physiology is 
related to a disease whereby a loss of function results in an important medical 
condition i'm think-, thinking of things like cystic fibrosis where the gain of 
a function is a problem for example a tumour tumour genes things like rasp 
found in a large proportion of human tumours and even when the normal function 
of a protein be-, could become a a 
medical problem i'm thinking there particularly of things like multidrug 
resistance both in bacteria and again in tumours so with that in mind we will 
briefly and it will be brief and this is essentially part of i guess your 
assumed knowledge we'll review the basic aspects of er protein structure and go 
on to some medically relevant examples so the basic er unit of protein 
structure is of course the peptide bond er a polypeptide er it's a sequence of 
amino acids that are joined through peptide bonds that represents the the 
primary structure of that protein the peptide bond is formed through the 
condensation of a carboxylic acid group from an amino acid and a amine group 
from an amino acid the er carbonyl oxygen and the nitrogen are electronegative 
and that effectively means that the peptide bond itself has extensive double-
bonded character it's rigid and planar it can't rotate but of course there's 
free rotation of the bonds on either side of that peptide bond and so the 
side chains of the amino acids could in theory form any number of angles in 
relation to that peptide bond i'm sure as you know in fact er there are 
relatively few angles that are allowed on either side of that peptide bond and 
most of the angles are not allowed because they're sterically unfavourable they 
represent clashes interactions between the er side chains of amino acids or the 
side chains of amino acids and the and the polypeptide chain and so the 
confirmations of the bonds on either side of the peptide bond can actually only 
form relatively few what are generally termed secondary structures the one with 
the largest scope is the is the beta sheet er i should say that psi and phi are 
of course the they're the names of the two bonds on either side of the peptide 
bond you don't really need to know that er but for a beta sheet those psi and 
phi angles suggest that the only the favourable energetic confirmation er for 
the side chains of these amino acids would be if 
they were essentially opposed on either side of the peptide bond and so they 
alternate on either side of the peptide bond er beta sheets er the that er 
opposition of er side chains allows for extensive hydrogen bonding between 
separate polypeptide chains that are in the beta sheet confirmation and i'm 
actually not really going to talk much more about beta sheets that's not 
because i don't think they're important er er or indeed have medically relevant 
examples it's just there isn't s-, time within this lecture to really go into 
beta sheet structures other than to describe er the Ramachandran conditions er 
obviously there are very pertinent examples indeed the transition between alpha 
helical structures and beta pleated sheet structures is pathological in various 
diseases such as Alzheimer's that's that ameloid transition the second set of 
allowed angles on either side of the peptide bond are essentially er offset by 
about i think about er a hundred-and-ten 
a hundred-and-twenty degrees and the only way you can conceptualize the 
arrangement of side chains in a polypeptide that are offset like that is if 
they're essentially rotating and that gives you the i-, notion that that would 
be a helix you can see there there's one small area that's allowed if van der 
Waals' radii radii are taken to be a little smaller and that's a left-handed 
helix and we'll talk about that er and generally er most amino acids fall into 
these Ramachandran rules Ramachandran was of course a Indian mathematician who 
who who er worked out these rules er for a secondary structure in the er turn 
of the s-, turn of last century now using both mathematical principles and er 
models of peptides the only amino acid that can be found in virtually any angle 
confirmation is er is glycine of course and that's because it's secondary er 
it's er sorry side chains of course hydrogen so most proteins contain alpha 
helix and indeed some are nearly all alpha 
helix and things like myoglobin and haemoglobin are predominantly alpha helical 
indeed the structures the parts of the protein that are not alpha helices in 
myoglobin and haemoglobin are involved in turning the molecule back on itself 
to give the very characteristic compacted shapes of those molecules so i'm sure 
that oh if anybody's struggling with their right-handed and left-handed helices 
that's what that's there for so this then is an actual alpha helix we can see 
that er the side chains are nicely arrayed to the outside of the helix on this 
side and on this side er that er it being it it being right-handed it pitches 
essentially from er er from the left towards the right upwards er in this 
particular helices you can see that there's essentially hydrophobic side chains 
on this side and er charged side chains on this side and this gives you an idea 
as to the orientation potentially of that helix in a protein obviously the 
hydrophobic side would be inward-facing 
the hydro philic side would be facing towards the aqueous environment you can 
see on the top there the how the er the er side chains essentially overlap and 
the er array themselves such they're approximately four residues apart as they 
go up the side of the helix so that's as i said there's approximately three-and-
a-half amino acids per turn and also you can see that the helices this 
arrangement of helices allows for hydrogen bonding within the chain so 
interchain hydrogen bonding and this helps stabilize the structure and it's why 
helical structures are so oft-, alpha helical structure energetically very 
favourable so if we look at the primary so the the the notion that there's 
periodicity or there appears to be certain periodicity in helices allows you to 
look at the primary structure and make predictions as to er what the secondary 
structure is most likely to be there is frequently periodicity in amino acid 
sequences er and certain amino acids are indeed 
preferred in helices yet others are not preferred in helices glycine isn't 
preferred in helices and proline as you know is a an amino acid that will 
essentially kink a polypeptide chain that won't be found in helices so this 
then demonstrates with that helices i was just showing you that the indeed the 
the polar residues er the sorry the non-polar residues are in the centre of the 
myoglobin molecule and the polar residues are arrayed on the surface aiding the 
solubility of myoglobin and in now allowing myoglobin and indeed haemoglobin as 
it's since it's very r-, closely related to myoglobin to er be in solution at 
relatively high concentration so this of course is what we are really going to 
talk about today myoglobin and haemoglobin this then shows the arrangement of 
the secondary structure wi-, in myoglobin which is predo-, predominantly alpha 
helical into the globin fold so that would be its tertiary structure and the 
tertiary structure is essentially an 
arrangement of eight alpha helices in globins er labelled A to H this was A B 
and C are behind here this is D this is E this is F this is G and this is H you 
can also see that in myoglobin it has a prosthetic group that's this red 
molecule here and this prosthetic group is essentially held in position in a 
myoglobin in a in a r-, in a a reasonable cleft or fold that helps protect that 
prosthetic group from oxidation and m-, and the prosthetic group haem of course 
is held in position by two histidine residues histidine E-seven and histidine F-
eight so it's the seventh residue in the er E helix and the eighth residue in 
the F helix you can see that the F-eight histidine appears to be quite close to 
the haem group the E histidine appears a little bit further away so the F- 
eight histidine is called the proximal histidine and the E-seven histidine is 
called the distal histidine so what we are going to talk about today of course 
is the binding of oxygen to myoglobin and 
haemoglobin we'll talk about the functions of myoglobin and haemoglobin er 
myoglobin acts as essentially as an oxygen store in muscle and it you'll see 
that its structure is entirely consistent with its function er whereas 
haemoglobin which is of course oxygen transport for transporting oxygen from 
the lungs to the tissues we'll talk about the factors affecting oxygen binding 
transport of carbon dioxide and P-H regulation when we consider the binding of 
oxygen to haemoglobin and indeed myoglobin we'll perhaps worth considering what 
atmospheric oxygen er the partial pressure axle atmospheric oxygen is around 
about a hundred-and-fifty m-, millimetres of mercury or twenty sometimes 
referred to as twenty pascals in other textbooks a significant proportion of 
that enters into the wet surface of the lungs providing of course those er lung 
surfaces are wet if they're not for whatever reason then you do not get 
efficient exchange between the atmospheric 
oxygen and the lung mucosa of the oxygen that dissolves into the lung mucosa 
the majority of that is taken up into the blood through exchange in the lungs 
oxygen then is er the blood is then circulated oxygenated by this circulated in 
the tissues and a relatively small proportion perhaps less certainly less than 
fifty per cent of oxygenated haemoglobin a relatively small proportion of the 
oxygen it carries is unloaded into the tissues carbon dioxide is picked up from 
the o-, from the active tissues and that's transported back via the blood back 
to the lungs and then back er in exchange back into the air that we breathe so 
one of the first factors that's going to affect the binding of oxygen to 
haemoglobin is the efficient physiology here at at the lung er circulation 
interface another more subtle perhaps er notion is that it all relies on the 
fact that atmospheric oxygen is what i said is up there and of course there are 
certain conditions when it's not for 
example altitude and that the lungs are er er functioning efficiently that the 
circulation is functioning efficiently you can see that of the oxygen that 
dissolves into the wet surface the majority of it is taken up by the lung by by 
the blood in the lungs so this is a fairly this is working to almost its 
maximum efficiency n-, at at this point and anything that affects that well it 
ultimately affects oxygenation of the tissues so the rate at which you breathe 
the so-called ventilation rate and the perfusion of the lungs I-E how much 
blood is passing through the lungs at any given time er must be matched that's 
called a ventilation and perfusion match and the ratio must be approximately 
four litres of breathing per minute and five litres of blood per minute passing 
through the lungs giving you a ventilation perfusion match of point-eight 
anything that impears impairs breathing such as a a crushed chest a deflated 
lung chronic lung disorder er somebody making 
the universal choking sign er will affect the amount of oxygen that's that's 
breathed that they're that they're able to obtain obviously if they have a 
problem with their blood flow like they're bleeding from a large artery in the 
leg will ultimately affect their oxygenation of their tissues but under those 
conditions i suggest you're more focused on the leg issue er at least initially 
and of course this relates to the A B C of first aid in many respects airway 
breathing circulation and any mismatch in ventilation or perfusion will 
ultimately lower the amount of oxygen in the arterial blood so that reveals to 
you that there's a problem with oxygen oxygenation so accepting that the lungs 
are working properly and there's the right the correct concentration of 
atmospheric oxygen we can then go back and and think about the binding of 
oxygen to haemoglobin and and myoglobin this is the haem er prosthetic group 
found in myoglobin and haemoglobin it's made up of the the carbony 
bit it's called the protoporphyrin and in the middle there is iron the ferrous 
iron that's iron two-plus the as i said the hydrophobic fold of myoglobin and 
haemoglobin protects oxidation of that iron if that iron does become oxygenated 
er oxidized it can no longer bind oxygen so in that and in that case it's that 
would be referred to as a met-myoglobin or a met-haemoglobin that's iron in the 
three-plus oxidation state the iron can make six coordination bonds it makes 
four with nitrogens that are in the pyrrole group it makes one with the pro-, 
with the nitrogen in the proximal histidine and the last one is for the binding 
of oxygen as i'll show you and essentially this molecule is planar in isolation 
so this then er puts the haem then in context of those two histidines er you 
can see here the proximal er histidine providing the s-, the fifth coordination 
point for the iron and oxygen is able to intercede between the the distal 
nitrogen in the histidine 
and that provides the binding site for oxygen to h-, to haemoglobin or indeed 
myoglobin and in this case in the presence of oxygen you can see that the haem 
remains planar and and i'll explain why that's important in a little while the 
other thing i want to point out is about er carbon monoxide isolated haem has a 
very strong affinity for carbon monoxide much more er er much stronger than it 
does for oxygen but haem in the presence of protein such as myoglobin or 
haemoglobin there are r-, the the differential between the affinities for 
carbon dioxide and oxygen is reduced such that it goes from twenty-five-
thousand times difference in isolated haem to two-hundred times different in 
the context of the protein so the protein plays an important role in hopefully 
limiting the effects of carbon dioxide the reason that that's important is of 
course is firstly carbon dioxide is the single most er single biggest metabolic 
poison it's far more potent and there are far more deaths 
from carbon monoxide than there are for any other metabolic poisons that might 
cyanide for example and carbon monoxide obviously actively competes for oxygen 
binding sites normally er the er when once carbon monoxide is bound to 
haemoglobin or to haemoglobin it will be referred to as carb-, 
carboxyhaemoglobin and there is a small percentage of carboxyhaem-, haemoglobin 
in our blood normally in smokers for example that percentage goes up to about 
fifteen per cent and around thirty per cent it's a medical emergency and around 
fifty per cent you're probably going to die so carboxyhaemoglobin is a 
significant problem because it's able to effectively reduce the oxygen ca-, 
carrying capacity of blood so now let's just think a little bit about this 
planar haem molecule this is the proximal histidine here's the planar haem 
molecule in the presence of oxygen in this form haemoglobin or myoglobin would 
be referred to as relaxed when it becomes deoxygenated the 
interaction between this nitrogen and the iron in the haem group is so strong 
that the haem tends to dome in to that er proximal histidine in this case the 
and that's deoxygenated form of haemoglobin it referred to as tense so the 
binding of oxygen to myoglobin or haemoglobin essentially represents a tense to 
relaxed transition and that tense to relaxed transition can be transmitted 
through the molecule because it is a shape change so we've talked in general 
about the binding of oxygen to haem and everything so far i've told you is the 
case for as it is for myoglobin as it is for haemoglobin but of course 
haemoglobin is actually a tetramer it has four globins so this is now a 
quarternary structure the globin fold of all four of the er subunits in 
haemoglobin is shown and you can see the four haem groups so oxygen is a er 
sorry so haemoglobin will bind four oxygens as opposed to myoglobin which will 
only bind one there you can see the 
doming in this particular haem group you can see the doming there of the haem 
group and of the i-, the iron being pulled into the proximal histidine adult 
haemoglobin there are there are three globin genes but there and there are 
multiple copies in your D-N-A er adult haemoglobin is predominantly so-called 
alpha-beta haemoglobin you always express a little bit of delt-, delta 
haemoglobin er during the first few months of life you switch from the fetal 
haemoglobin to these adult forms so obviously fetuses fetal expression is of 
the H-B-F and an adult's a little bit of H-B-A-two and H-B-A sometimes also 
referred to as H-B-A-one so now we understand a little bit more about the 
structure of haemoglobin and myoglobin how can we relate that to their 
functions on the r-, on the left-hand side we have the s-, degree of saturation 
oxygen saturation of the proteins and er partial pressure of oxygen along the 
bottom the green line represents the binding of oxygen to myoglobin and it 
shows a 
simple hyperbolic relationship entirely consistent with a single binding site 
and its responsiveness to changes in oxygen pressure are consistent with its 
role as a oxygen storage protein in muscle myoglobin is rapidly saturated over 
a relatively small concentra-, change in concentration and will remain 
saturated as a tissue a muscle's demand for oxygen increases er some oxygen can 
then be released from myoglobin in order to support the metabolism in active 
muscle this is er quite different to the curve that you see for haemoglobin 
which is sigmoidal in shape consistent with a multiple binding site which we as 
we know is four this type of er curve a-, reflects what's called cooperative or 
politi-, positi-, 
nm0442: allosteric binding so oxygen here acts as an allosteric regulator of 
the binding of oxygen to haemoglobin the way to conceptualize that is this here 
at the very over a relatively wide concentration range at er here at the low 
end of er haemoglobin's 
responsiveness to oxygen it's very reluctant to give up its oxygen as oxygen er 
partial pressure increases there's a rapid increase in haemoglobin's ability to 
bind oxygen the only way you can s-, conceptualize that is if the subunits are 
cooperating in their binding of oxygen to haemoglobin the probably the better 
way the best way to think of it is as they release is as haemoglobin release 
oxygen as it releases oxygen it becomes progressively harder for haemoglobin to 
release further oxygen and of course here fully saturated the haem groups are 
relaxed and here as they're deoxygenated they become tense so the binding of 
oxygen to haemoglobin er exhibits positive allosterism it is cooperative the 
cooperativity essentially represents communication between the subunits the er 
quarternary structure i showed you showed that the subunits are in very close 
proximity and there are interaction faces between all of the four subunits so 
the binding of oxygen for example here 
perhaps in the red subunit can be transmitted to the other four subunits so 
they're in close proximity and that's because there's a change of shape at the 
haem group and that can be transmitted with a much larger change of shape at 
the interactional face this er change in terms of oxygenated and relaxed 
haemoglobin tends to close a pocket that form-, that can form in the middle of 
the four subunits in a deoxygenated form this pocket tends to be a little bit 
more open and we'll see the relevance of that in a minute so oxygen represents 
a positive allosteric regulator of the binding of oxygen to haemoglobin and 
there are also some negative allosteric regulators of the binding of oxygen to 
haemoglobin of course i should remind you of course that myoglobin is a monomer 
so there is no allosteric regulation of the binding of oxygen to myoglobin this 
i'm sure you'll recognize is a little bit of glycolysis which i all medical 
students are always delighted 
to see glycolysis [laughter] and er in the erythrocytes there is a small side 
er reaction that's able to convert one of the intermediates of glycolysis to 
one-three-bisphosphoglycerate into two-three-bisphosphoglycerate it's 
discovered by this guy Rapoport and Luebering and it's called the Rapoport-
Luebering shunt not as somebody put in their exams last year the Freddie 
Ljungberg shunt [laughter] two-three-bisphosphoglycerate is electronegative and 
it is at very high concentration in erythrocytes it's approximately equimolar 
with the concentration of haemoglobin in erythrocytes so you can see glucose as 
it enters into glycolysis essentially all the intermediates in glycolysis 
there's very little of those glucose enters into glycolysis in essentially in 
erythrocytes of course is converted to lactate because they have no citric acid 
cycle no mitochondria and so on the only other intermediate 
is two-three-bisphosphoglycerate formed by the the shunt and the role of two-
three-bisphosphoglycerate is effecti-, is e-, to effectively lower the affinity 
of haemoglobin for oxygen this is a little bit schematic in the absence of er 
bisphosphoglycerate this this curve actually would shift be shifted quite quite 
a little way to the left in relation to the normal haemoglobin in erythrocytes 
so in the absence of er in the absence of two-three-bisphosphoglycerate 
haemoglobin would be quite reluctant to give up its oxygen at all obviously as 
this would be a problem for the delivery of oxygen to tissues and ov-, and 
clearly we have adapted to use two-three-bo bisphosphoglycerate to enable us to 
moderate the release of oxygen er in the active tissues the two-three-
bisphosphoglycerate is is is binding site is found in the centre of the 
haemoglobin er tetramer it's predominantly formed by interactions with some 
alpha chain interactions and predominantly beta chain interactions er and these 
are positive residues electropositive residues like lysine and arginine and 
histidine two-three-B-P-G combined to the deoxygenated form of haemoglobin 
helps stabilize er the dexoygenated form and helps release oxygen into the 
tissues a clear demonstration of the importance of two-three-
bisphosphoglycerate is the fact that there is no two-three-bisphosphoglycerate 
binding site in fetal haemoglobin so the gamma chain in er fetal haemoglobin 
does not have those electropos-, positive residues so that binding site isn't 
present this means that fetal haemoglobin has a higher affinity er for oxygen 
than does the maternal haemoglobin and that ensures that oxygen will flow from 
maternal haemoglobin to fetal haemoglobin one can imagine other scenarios where 
the level of two-three-bisphosphoglycerate would be moderated in order to 
increase the supply of oxygen to the tissues for example at altitude one of the 
adaptions to altitude is to increase your levels of 
two-three-bisphosphoglycerate and that helps unload oxygen in the tissues 
chronic lung disease is another case in which you see increases in two-three-
bisphosphoglycerate concentration as the body's attempting to compensate for 
poor oxygenation and to deliver as much oxygen as possibly can to the tissues 
this has to be a temporary solution and in people with chronic lung disease it 
it itself perpetuates the problem since they increase their two-three-
bisphosphoglycerate and it becomes progressively harder then to reoxygenate 
your haemoglobin in the lungs does everybody understand the role of two-three-
bisphosphoglycerate is there any divers in the room any divers scuba people 
come on there must be one or two you if i give you the choice imaginary choice 
two-three-bisphosphoglycerate small white powder or air and you're going diving 
which are you going to take 
sf0443: 
it's obviously a trick question isn't it 
nm0442: no it's not a trick question [laughter] 
sf0443: 
nm0442: the air you're going to take the air 
sf0443: yeah 
nm0442: two-three-bisphosphoglycerate is not an oxygen substitute okay one of 
the favourite answers of medical students think of the diver give him the 
option of the white powder or the air and see which one he takes nobody takes 
the two-three-bisphosphoglycerate it is a negative allosteric regulator of the 
binding of oxygen to haemoglobin it is not an oxygen substitute good there are 
two other negative allosteric regulators of the binding of oxygen to 
haemoglobin they are hydrogen and carbon dioxide and as you can see here a d-, 
a decrease in P-H effectively reduces the affinity of haemoglobin for oxygen 
and that helps unload oxygen in active tissue since in 
active tissue the concentration of hydrogen and carbon dioxide is high both 
these molecules do so by interfering with cross-bridging that interaction 
between haemoglobin subunits carbon dioxide can interfere with cross-bridging 
firstly because it actually of course can in solution can increase the hydrogen 
ion concentration as we'll see in a minute and secondly because i-, it can bind 
to haemoglobin it it doesn't bind at the ion so a certain amount of carbon 
dioxide is transported by haemoglobin but is transported covalently attached to 
free amine groups so that's called carbaminohaemoglobin and that's different 
from carboxyhaemoglobin which is carrying carbon monoxide at the ion so 
carbaminohaemoglobin carrying carbon dioxide obviously interferes with cross-
bridging interferes with oxygen binding and hydrogen ions interfere with cross-
bridging communication between subunits and can lower the affinity of 
haemoglobin for oxygen so let's just consider a 
little bit the transport of carbon dioxide carbon dioxide and water can be 
combined in the erythrocyte using the a-, enzyme carbonic anhydrogena-, 
carbonic anhydrase to produce carbonic acid which rapidly diassociates to form 
a carbonate ion and hydrogen the majority of carbon dioxide produced in tissues 
is transported in the form of carbonate once it's made in the erythrocyte it 
passes out through the erythrocyte in a specific transporter and i-, er present 
in the plasma a relatively small proportion is transported back actually 
covalently linked to er haemoglobin and interestingly of course the covalent 
link of carbon dioxide to haemoglobin also lowers D-P-H raises the hydrogen ion 
concentration just to remind you of course carbon dioxide protons all their 
equivalents are of course the major metabolic end points when i say equivalents 
i'm thinking about lactate lactate or ketones what have you so we come back we 
f-, we return 
to the lungs and consider now the binding of oxygen to haemoglobin in the lungs 
here i've represented haemoglobin carrying a proton which is released as oxygen 
binds this proton can drive the carbonate equilibrium to the left pushing 
carbonate back to carbonic acid and causing the the evolution of carbon dioxide 
in the lungs oxygenation of this species is reducing the concentration of this 
species in the blood and that helps drive this equilibrium to the left to 
release the bound carbon dioxide that's bound to haemoglobin so then the lungs 
of course because oxygen concentration is high favours oxygenation of 
haemoglobin so that's shown on the left sort of schematically the erythrocyte 
the alveolar surface incoming oxygen the shift in the hydrogen ions so-called 
isohydride shift and the driving of carbon dioxide out of the erythrocytes and 
into the alveoli and into the air we breathe out and exactly the opposite is 
true in active tissues in active tissues carbon 
dioxide and hydrogen are are concentrations are high they're taken into the 
erythrocyte converted to the carbonate ion the carbonate ion is transported out 
by er er a chloride carbonate antiporter it's called band three in erythrocytes 
you'll learn a bit more about transporters in the next couple of weeks i think 
there's the isohydride shift that helps to drive oxygen off haemoglobin and 
drive it into the tissues where it's needed obviously all this er movement of 
hydrogen ions essentially means that the P-H in blood has to be very tightly 
regulated normal physiological pa-, P-H is in a very narrow range and any 
factors that t-, er take the normal physiological P-H outside this range can be 
dager-, dangerous indeed deadly you ca-, if you would look up er you can look 
up things like er acidosis in newborn children or er er premature children and 
you can see the you'll be able to read about the effects of acido-, that 
acidosis has on both the physiology er the long term development 
of children their mental acuity and so on so regulating P-H is very very 
important and of course the main system that does it is the one we've just 
described it's the bicarbonate buffering system this represents about seventy 
per cent of the buffering capacity of blood phosphate also can be involved in 
er buffering and indeed of course the various proteins can do a little bit of 
buffering but of course this system is open it's open at this end because it's 
open in the lungs the carbon dioxide the equilibrium's open at that end the er 
equilibrium's open at the proton end because protons are the major metabolic 
end points as indeed is carbon dioxide so we can relate the control of blood to 
the way in which we are respiring if we're hyperventilating perhaps panicking 
about the molecules exam [laughter] then we're breathing deeply and rapidly 
that will force carbon dioxide out of the lungs draw this equilibrium to the 
left pulling protons 
in and the P-H of the blood will essentially become more alkali so in-, yeah so 
it so hyperventilation is towards the alkali poor ventilation chronic 
obstructive pulmonary disease is going to keep carbon dioxide deep in the lungs 
and force the equilibrium to the left and the hydrogen ion concentration will 
increase so hyperventi-, hypoventilation is going to be giving you acidosis and 
hyperventilation is giving you alkalosis this of course the realization here is 
of course that the the lungs represent a very major control of P-H so here's 
respiratory alkalosis caused by hyperventilation simple treatment rebreathe or 
administer carbon dioxide conversely re-, respiratory acidosis caused by 
inadequate breathing unable to exchange the carbon dioxide in the lungs er have 
to administer more buffering probably I-V so the important point here is that 
because those equilibria are open at 
either end er respiratory changes in P-H are related to metabolic changes in P-
H so there are various much more common conditions that for example 
uncontrolled diabetes diarrhoea overdose of aspirin where or indeed prolonged 
heavy exercise hec-, exercise where you become metabolically acidotic you 
produce a lot of acid equivalents and that interferes with that carbonate 
buffering system and it overloads it at the right-hand end and so you go into 
deep rapid breathing you hyperventilate to blow off carbon dioxide what you're 
trying to do is pull hydrogen ions in from the metabolic acidosis and blow 
carbon dioxide off so respiratory alkalosis is linked to metabolic acidosis and 
therefore a m-, a respiratory alkalosis although is a condition itself may be 
caused by metabolic acidosis similarly the opposite is true if you're prolonged 
vomiting you lose a lot of hydrogen ions from the stomach you become er and you 
go into metabolic alkalosis then your breathing becomes 
shallow and infrequent as the body attempts to compensate for that by holding 
on to the carbon dioxide forcing the equilibria to the right and increasing the 
hydrogen ion concentration to combat the alkalosis in the blood okay so we've 
talked about the transport of oxygen we've talked about the transport of car-, 
carbon dioxide and we've even talked a little bit about respiratory control of 
P-H i've talked about acidosis and alkalosis in relation to the lung the other 
major organ which i'm not going to talk about that controls P-H is of course 
the kidney so clinical correlations other clinical correlations perhaps 
haemoglobin can become glycosylated in blood this is a spontaneous er reaction 
between haemoglobin and glucose and in uncontrolled diabetes when glucose 
levels are elevated in blood glycated haemoglobin acts as a measure over the 
previous three three or four months er acts as a measure of the level of 
control of diabetes for example if er a diabetic 
hasn't been following the insulin regime their glucose concentration will have 
been fluctuating wildly in their blood and gluc-, and the glycosylated 
haemoglobin acts as a marker for that we've already talked about the role of 
two-three-bisphosphoglycerate stored blood of course the two-three 
bisphosphoglycerate rapidly er degrades so it nus-, needs to be added to stored 
blood if it's going to be transfused and we've also talked about the 
physiological er conditions that may alter the level of two-three-
bisphosphoglycerate what we haven't talked about at all are haemoglobinopathies 
such as sickle cell and the related thalassemias they are relatively rare and 
there are many of them they all relate to either alterations in the globin 
structure or indeed er a loss of expression of a globin gene thalassemias p-, 
vary in their er severity from relatively mild to relatively severe er and this 
would be an area i suggest you have a brief look at if you get the 
time right hello oh good well that has taken nearly well just over half the 
lecture i thought we'd just have a little break now and do something a little 
bit different 
sf0444: so obviously they just brought somebody in unconscious 
sf0444: and Malucci being a bigot just ask first and then he completely ignores 
what he said 
sf0444: heart attack 
sf0444: so Malucci's really caught up in the he's 
sf0444: and now he's just trying to pressure the doctor into making his 
decision 
sf0444: they've just lost the patient 
sf0444: now in the scene 
sf0444: and then he's still stuck on the fact that it's drug-related though 
sf0444: so they've started giving a clot-buster to somebody who's completely 
bleeding out so now he has no and he's haemorrhaging and that's the i'm in big 
shit look 
nm0442: 
round of applause for namex there well done ooh get rid of that okay so the 
classic differential on M-I is dissection the aorta the aorta splitting and if 
you misdiagnose your M-I you give a clot-buster and they bleed into the chest 
because they're dissecting and it was the clot that was holding them together 
and as er Carter the unstoppable sex machine spotted [laughter] er this guy's a 
Marfan and a Marfan is er a collagen or it's actually a collagen-related 
disease he has Marfan's syndrome and Marfan's syndrome is a defect in collagen 
and frequently er pathologically these people suffer from vascular problems and 
various other presentations of a a collagen disease so what we're going to talk 
about now is collagen so in everything i've kind of told you about haemoglobin 
and about helices when it comes to collagen you 
begin to throw all these things out of the window the first thing is that it 
has er essentially a repeating structure of of essentially three amino acids 
it's a gly-X-Y as it says there gly-X-Y motif and X and Y are either proline or 
hydroxyproline lysine or hydroxylysine mainly er it's found in of course 
tendons cartilage bones on the surface on the articular surface of bones even 
in the eye er the micrograph there just gives you an idea of the structure of 
collagen fibres and we see this kite quite characteristic er banded pattern 
that's seen in obviously only in fibres and then at the end there this cross 
section you can see how they represent bu-, fibres of er bundles of fibres 
bundled together there are it says at least ten there are about thirty 
different types of collagen some form fibrils some s-, form mal-, small fibrils 
called microfibrils some are associated with fibrils and are a little bit 
fibrillar-like but also have interruptions in their fibrillar 
structure and some just form network meshworks so there are fibrillar types 
microfibrillar types non-fibrillar types and fibrillar associated collagens 
with interrupted triple helix facit okay for those of you are from Essex that's 
not fuck it [laughter] okay and the structure is completely different it's a 
left-handed helix it is a very tight turning helix and it's it's left-handed 
and tight turning because of the residues in there proline helps er the tight 
turn as does hydroxyproline glycine has virtually no side chain so that allows 
the turn to be very tight and a single collagen left-handed helix is wound with 
two other left-handed helices to form a right-handed helices er a coiled coil 
and coiled coils of course structurally are very strong that's not to say that 
er any strong protein structure has to be helices because there are plenty of 
examples of strong protein structures that are beta sheet and silk fibroid is a 
very good example of that 
but nevertheless coiled coiled type structures are structurally very strong so 
i'm just going to briefly review the biosynthesis and post-translation of 
collagen and then we're going to go through it in a little more detail in 
subsequent slides but basically of course the M-R-N-A is translated and it is 
both translated and secreted into the E-R in the E-R the lysines and prolines 
become hydroxylated by specific hydroxylases some of the hydroxylated residues 
will subsequently become glycosylated so there's O-link glycosylation on those 
hydroxylated residues others will be involved in promoting hydrogen bonding and 
it the hydroxy residues are involved in hydrogen bonding and also indeed in 
cross-linking as we'll see further along there's also some end-linked 
glycosylation of of collagen and the ends of the collagen molecule are 
disulphide bridged to form a sort of globular structure so there are several 
post-translational modifications that occur in the 
E-R the hydroxylation the glycosylation and the formation of disulphide bonds 
three collagen helices are then wound together and this winding involves the er 
r-, arraying these collage-, these s-, sorry globular gr-, er modules at the 
end of the collagen fibre they help bring the three collagen fibres together 
and promote the winding of three collagen fibres glycosylation in amongst the 
wound areas helps er interrupt the helices hence interrupted triple helix go-, 
so glycosylation plays an important role in er interrupting the triple helix it 
also plays a role in maintaining the solubility of collagen so it's a the er 
carbohydrate molecules help attract the water this then is secreted out of the 
cell and the secretion of such a large molecule is not an insignificant task 
the globular portions will be removed leaving the mature fibre with a degree of 
interruption in the triple helix or not and some of the re-, residual hydroxyl 
groups particularly the ones at 
the ends of the mature fibre are involved in cross-linking this fibre to 
another fibre and that cross-linking er is evidenced by the banded pattern that 
we see in collagen in fibrillar collagens so in a little more detail then er 
this exe-, example for proline there are specific er hydroxylases in the er E-R 
and they're vitamin C requiring the absence of vitamin C er results in improper 
hydroxylation of proline and lysine and vitamin C deficiency is of course 
scurvy i quite like this little description of scurvy from er Jacques Cartier 
who er discovered Canada fifteen-hundred like the last bi-, bit the best their 
mouth became stinking their gums so rotten that all the flesh did fall off even 
to the roots of the teeth teeth which did also almost all fall out course in 
retrospect he didn't know that they were on the Atkins diet [laughter] 
[laughter] so first key point in er collagen biosynthesis you need vitamin C 
the sp-, the the 
residues glycine hydroxyproline and proline permit the association of the three 
chai-, the three chains together and they can be become intimately interwoven 
here one of the residues er has been mutated in silico in the computer to an 
alanine and that drives the chain apart in this area so a simple mutation in 
collagen can drive the chains apart and weaken the overall collagen structure 
this is of course the basis of osteogenesis imperfecta where there is a 
mutation of a glycine in s-, mi-, most of the time it's to a cysteine it occurs 
in one of the major coloni-, collagen types there are sev-, because there are 
three three collagen genes there are multiple variations into the presentation 
of osteogenesis imperfecta normally they of course will have brittle bones as 
the name suggests osteogenesis is generation of bones imperfecta imperfect it 
is relatively rare and in certain cases can be absolutely lethal give you an 
idea of what sort of X-ray of what a p-, a baby with 
osteogenesis imperfecta looks like you can see these multiple fractures here 
the sort of thickening and the thinneni-, thinning of the bone these you can't 
see the ribs very well but the ribs are v-, quite characteristically in this 
sort of er this sort of er appearance on X-ray so it's quite a severe disease 
it's also some s-, frequently collagen disor-, disorders can be picked up in 
the whites of the eye the so-called sclera of the eye they have a s-, slightly 
blue or greyish tinge because of the collagen that's in the er the whites of 
the eye and in er patients with osteogenesis imperfecta there's less collagen 
in the eye and a pigment from behind the e-, the eye shows through and so they 
have an apparent blue tinge to the sclera the part of the structural integrity 
of collagen is through hydrogen bonding between those hydroxyprolines er and er 
glycines and proline residues that gives cross-chain or interchain hydrogen 
bonding it helps stabilize the hae-, helix and contribute 
to the mechanical strength that's seen in collagenous er tissues like tendon 
here we can see a little more detail on the the role of this globular domain it 
helps bring the three chains together allows them to twist together to form the 
mature tropocollagen and then these are removed in the extracellular space by 
an enzyme called procollagen peptidase a deficiency in procollagen peptidase is 
characterized by a syndrome called Ehlers syndrome Ehlers Danlos syndrome heard 
a little bit about this in the genetics lecture because Ehlers' very complex 
can have complex inheritance it's quite char-, characteristic hypermobile 
joints and very stretchy skin 
nm0442: er and like many of these er collagen and collagen-related diseases the 
prognosis may be shortened because of vascular complications in the heart or in 
the major vessels once the mature collagen fibre has been secreted into the 
extras-, extracellular space it's cross-linked it's cross-linked at the en-, at 
the ends here free hydroxyls from l-, er free hydroxyls free hydroxylysines or 
indeed lysines are covalently linked across these triple helices this gives you 
an idea actually this is a lysine cross link to form a so-called shift base you 
don't really you just need to know that that's a covalent link you don't really 
need to worry about the f-, the fan-, fascinating structure of a shift base er 
nevertheless there is another disease associated with the improper cross-
linking of collagen and that's called lathyrism inhibition of lysyl oxidase the 
cro-, the collagen cross-linking enzyme by found this compound's found in sweet 
pea so although sweet pea of course is a very good source of vitamin C it's not 
necessarily the best cure for scurvy because actually what you do is you 
inhibit the lysyl oxidase okay and again a collagen disease where dislocations 
of the joints and vascular problems are associated with the disease all of 
those diseases represent loss of function 
but gain of function or deposition of collagen can be le-, can be equally as 
dangerous it's referred to medically as fibrosis in exa-, for example in a 
medical student liver being constantly abused by alcohol scarred collagen is 
busily being deposited in that gentleman up there who's [laughter] busy 
partying every night and er that can result in impaired liver function 
similarly in the heart fibrosis of the heart tissue is going to result in 
severe er reduction in the efficiency of the heart in stroke volume and so on 
and that and that er again serious er complication lung fibrosis is quite 
interesting and and of course lung fibrosis is going to dramatically reduce the 
oxygen uptake the oxygen uptake ability of the lungs and lung fibrosis is 
frequently caused er in it's frequently found in patients in long term stay in 
hospital and can be induced indeed quite rapidly by certain drug treatments as 
well so you can you can treat patients with certain drugs and 
all of a sudden you induce lung fun-, fibrosis and it's a very serious problem 
so both loss of function and gain of function of collagen are serious medical 
issues in the main we've talked about these fibrillar collagens and the er the 
er notion that they're they're of course involved in ligaments tendons in the 
architecture of joints er that they are in in in those cases they're complex 
molecules they contain fibrillar collagens microfibrils they contain collagens 
that are is-, just there to be associated with the fibril other collagens and 
du-, generally these collagens will be much more heavily glycosylated form 
branch structures they form meshworks that are involved in the formation of 
basement membranes which endothelial layers grow on the when on which 
fibroblast layers will grow right well should just say as well of course in in 
this case the extracellular matrix is not just collagen it's associated with a 
lot of other proteins proteoglycans er glycoproteins and the 
various elastins and it's the elastin gene which there's only one er er gene er 
one elastin gene that's mutated in Marfan it's similar to collagen it has this 
glycine and proline rich sequence has er more valine in it it's essentially 
very hydrophobic it it er it's le-, much less post-translationally modified 
than is collagen it has very little secondary struc-, recognisable secondary 
structure perhaps that's consistent with its er more elastic properties it is 
cross-linked in the same way as collagen is by the lysyl oxidase and it's 
mutations in that fibrillin gene that make up elastin fibres that are 
responsible for the Marfan syndrome and quite characteristically the Marfans 
are tall they have er long limbs arachnodactyly which is spidery long fingers 
and the thing that er alerted Carter was the pec-, pectus excavatum which 
essentially means a slightly sunken chest so in just about an hour we have 
reviewed protein structure we've talked about the 
binding regulation of the binding of oxygen to haemoglobin and myoglobin and 
we've even watched telly and done the collagen diseases you will notice at the 
end of your lecture handout that there are a few sli-, there are a few slides 
i've included about er the molecular basis of the contraction of muscle 
something that we don't have time to cover in this series and something i 
suggest you use your hour now to have a little brief look at and familiarize 
yourself with in particular the basic er apparatus that's involved in skeletal 
smooth and cardiac muscle that regulates the contraction and even a little bit 
on how to recognize what a skeletal muscle cell looks like compared to a smooth 
muscle cell compared to a cardiac muscle okay so we're done for the the groups 
that have got to go i suggest you go if there's anybody who wants to come and 
ask me something come down when when the first group have got away