so everything I've talked about up to now has been focused on secrete proteins those are soluble proteins that are eventually dumped outside the cell and what I want to do is even though many of those rules also apply to transmembrane proteins what I wanted to do is kind of give you a sense of the diversity and of the different types of membrane protein topology is how they can insert into a membrane and then really focus on the experiments that can be used to determine how a protein loops in and out of the membrane and kind of what rules determine the topology now a lot of textbooks a lot of classes go through a lot of detail about the various kinds of sequences that can determine membrane protein topology I'm not really gonna go into that because in my opinion the key thing is to understand how you can figure it out in kind of the underlying logic because then it's easier to create exam questions in my opinion that actually test knowledge as opposed to your ability to spew back huge amounts of detail so just to give you a sense of kind of how diverse transmembrane proteins can be so here's the membrane we can have a transmembrane protein where the amino terminus is inside the lumen and the carboxy terminus is outside so this is the lumen and this is the cytoplasm conversely here's the transmembrane sequence originally you can also have topologies where everything is flipped and then you can even have things where the amino terminus is the actual transmembrane domains itself and it's anchored in the membrane so those are some simple things but then you can have multiple membrane spanning proteins so that's a protein that spans at multiple times and these can get quite complicated so for example CFTR the gene that cut is responsible for cystic fibrosis it has 12 different membrane spanning domains so it loops back and forth across the membrane 12 different times in fact that topology was somewhat controversial for many years and it took a lot of experimental work to figure out exactly which side which parts of the protein are on which sides of the membrane which is really important for understanding how different disease-causing mutations actually work and these are just the simple proteins that I'm diagramming here you can also have other types of linkages and in this case I'm drawing one that has a GPI linkage which is a completely different mechanism for anchoring something in the membrane so these can get quite complex and what I wanted to focus on rather than kind of a lot of the sequences that determine how the different transmembrane domains get read out by the 661 channel because a lot of that is like still relatively poorly understood what I wanted to focus on are the experiments or the assays to determine membrane topology and by topology I mean the nature in which it loops back and forth across the membrane so so the first classic essay that people had was proteolysis so you can imagine you have a membrane protein and let's just put this on the cytosol this is the lumen and it loops back and forth now if you treat that with a protease what happens is the protease is out here on the outside like a little Pac Man coming in to chew up the external domains and so what you end up with is just the fragments that are protected from the protease so a simple way let's see if I've drawn this correctly so all these external domains which I'm going to just draw in these were all chewed up by the protease and so what you have are 1 2 3 4 different protein fragments and so if you have antibodies to different parts of the protein you can begin to do this kind of molecular Sudoku puzzle of trying to figure out what when I treat with a protease what fragments do I have remaining and what does that mean in terms of which parts of the protein are exposed to the cytosol oxide and which parts of the protein are on the luminal side so that's a classic way of figuring out by protease accessibility or sensitivity and it's you know it's very time consuming it requires a lot of reagents and it's it's difficult to kind of go from the pattern of bands that you see in working with but it is a classic way of doing this now the second type of assay would be involved tagging the protein with an enzyme and I'll show you why that's useful and it for for this example I'm going to use alkaline phosphatase which I'm going to abbreviate ap you can you'll see in the literature and other textbooks that people use other types of enzymes ba galactosidase basically any enzyme that has a substrate or artificial substrate that can be turned into some sort of from clear to some sort of color or some sort of fluorescent material so anything that's an easy visual assay for enzyme activity now why do you want to do something like that so what people do is they say okay we've cloned this enzyme we have alkaline phosphatase enzyme and we have our protein of interest alright and it has let's say three believed to be transmembrane domains right and at the C terminus we're gonna stick how come fall so taste now this is a gene fusion so basically you make your protein and then you remove the stop codon and you drop in alkaline phosphatase so it's one continuous polypeptide it's your protein of interest and at the C terminus you have alkaline phosphatase so you could imagine two different scenarios for this protein if you were to do in-vitro translocation scenario one is the amino terminus is on the outside it loops back and forth and the alkaline phosphatase at the C terminus ends up on the inside so that's scenario one scenario two I'll just make it a different color to make it clear is that the amino terminus is on the inside and it loops back and forth three times because there's three transmembrane domains and the alkyne phosphatase ends up on the outside so these are two possible topologies of this three transmembrane domain protein however in scenario one so let me just make this an arrow to make it clear scenario one the alkaline phosphatase is inside the vesicle so what does that mean it means there's no access to substrate so let me just make this you know so if the substrates all floating around out here alkaline phosphotase can't act on it and change it to a color so so everything remains clear so that's scenario one so what happens with scenario two so with scenario two what happens is the alkaline phosphotase is outside the membrane and what that means is now the enzyme substrate can interact with the enzyme and let's just say when it interacts with the enzyme all of a sudden it starts turning or generating a blue color so the substrate changed color it changes color and so you can tell based on where the alkaline phosphatase ends up whether it's inside or outside and if you have an idea of how many transmembrane domains you are you have in the protein you can figure out the topology based on where the c-terminus ends up so those are the assays and I use them in a lot of different problem sets in terms of figuring out like given a specific scenario or a set of experimental results can you figure out what the actual topology of the protein is but there are some standard rules on how the membranes insert or organize within the membrane and so I'm going to walk you through a few of those just to give you some context some of these are just logical implications of how membrane proteins traverse the membrane but it's always good to just like draw them out into the open so it's very clear so so the first thing and this is really useful is if you have an odd number of transmembrane and I'm a breathing that abbreviating that TM transmembrane domains then the amino and carboxyl terminus the ends of the protein are on opposite sides of the membrane and you can kind of see that here so if I drew a membrane let's just extend it out a little bit so if I go one two three and and see our on opposite sides and if I go I can start on the opposite side one two three four five pretty much no matter what I do and it just baked into if you cross the if you if you cross the membrane three times you end up on the opposite side of where you started so conversely an even number of transmembrane domains then the amino and carboxyl end 's are on the same side of the membrane so what does that mean well that means that's one of the reasons that alkaline phosphotase si is so powerful is that if I can figure if I know how many transmembrane domains there are or I have a good idea and I know where the C terminus is because based on what if the alkaline phosphatases inside the membrane or or outside the vesicle then I can essentially draw the entire molecule and how it spans the the membrane so that's why this particular rule it's more an observation and I'll just quickly draw an even number just to confirm just to give you a sense six give you a sense of how this rule applies so it's it's more just a kind of a mathematical observation of how odd and even crossings work isn't helmets just a basic rule of topology but when you start combining that with the experimental assays you can really it becomes very powerful so a second rule and this is more an empirical observation and so a lot of professors kind of key in on this because it's something kind of a little bit of trivia that people like to play with is that charge so charged amino acids determine the orientation of the transmembrane domain and what do I mean by that it means that if you have positive charges is always on the cytoplasmic or cytosolic face so what does that mean so obviously a protein it has a bunch of you know most proteins that are gonna have hydrophobic residues for the transmembrane domain is because this has to stay embedded in the membrane but then you can have you know a variety of polar nonpolar positive negatively charged residues but the observation is that if you look at a protein once it's already embedded in the membrane so here's the membrane I'm just gonna draw it with a different color so if you imagine this and this is cytosol ik and this is luminol if you were to see if you were to guess where you might see a cluster of positive charges those positive charges would be found on the cytoplasmic side of the transmembrane domain so if you were to draw this out as a linear sequence so assuming that this is n and this is C what you would see is let me just so this was for transmembrane domains one two three four I might expect to see positive charges could be here a cluster of them they don't have to be that's always the thing is it's not something you always see but if you saw a cluster positive a charged amino acids on one side of a transmembrane domain those are almost always found on the cytosol like face so I could see them saij no that would be here so if you follow this around so you start an amino terminal side there's this is cytosol ik this is lumen cytosol ik lumen cytosol so you can eventually see oh I'm gonna see some positively charges maybe here and then maybe on the far side of that one so so if someone gives you a Seaquest and says that there's a cluster of positive charges on this side of a transmembrane domain where would you expect it to be you would expect that that cluster would be facing the cytoplasmic side so it's a useful trick it's also something a lot of professors put into prom sets and various things so it's good factoids to know and then lastly I'm not gonna get into the details of these sequences I'm just gonna let you know that they exist but in order to generate these complicated membrane protein topologies where they span and loop multiple times as you might imagine figuring out how to spool the spaghetti so that loops across the membrane 12 times with the correct portions on the correct side of the membrane it's a little bit challenging so there's a whole set of what are called tapa genic sequences and they're relatively poorly understood but they're the ones that instruct the sex 61 channel what to do like to continue translocation to stop translocation to allow that transmembrane domain to exit sideways and to diffuse laterally into the membrane so you know there's a lot of information content but they're poorly understood and eventually hopefully we'll understand them well enough that when given a primary amino acid sequence you could predict the topology of the protein with a high fidelity but two sequences that are just worth knowing about their existence because they often crop up our signal anchor sequences and stop stop transfer anchor sequences so why why mention these signal anchor sequences instruct the transmembrane domain stays in the channel while translating so what that allows it to do is like you know if you have a huge amount of sequence after the transmembrane domain you don't want that transmembrane domain to continue on you don't want to spool through the channel and if you let it go lateral it might start to thread into the channel and create an additional fold so what often happens is that transmembrane domain when it has a signal anchor sequence it anchors it and then the ribosome essentially detaches from the channel and just continues translating the rest of the cytoplasmic domain in contrast these stop transfer anchor sequences instruct the transmembrane domain to exit the channel and that's really important so if you're making 12 different trans membrane spanning regions so at a certain point you can't you can't cram 12 different trans membrane spanning peptides into the channel so there comes a point where you do it you have the transmembrane domain in the channel but when you're queuing up and you're ready to move on to the next transmembrane domain this signal see this stop transfer anchor sequence allows it to traverse laterally and then it can start working on the next set of loops so those are both really important type of genetic sequences again I'm not going to go into it but it's important for you to understand that there are these signals that tell it how to loop in and out and they're all being read by the channel