How do you store membrane proteins?

We're producing some membrane proteins and they aren't amenable to freeze thaws even when we add glycerol. The proteins are solubilized in detergent above the cmc so they should be in micelle form in solution. Currently, they are being held at 4C. I'm curious what is the best way to store them for long term use?

I work with membrane proteins and in my experience some proteins can be frozen and thawed without any problems, other proteins just don't like that. It's linked to the stability of the protein. If you really need to freeze the protein you may think about playing around with different detergents to see if you can improve the stability of the protein.

General rules: - freezing membrane proteins at -80°C without glycerol - avoiding protein freeze-thaw cycles, making aliquots before freezing - keeping them at 4°C only for short term use - after concentration always spinning the protein (10 min at 100k for example) to precipitate aggregated material. Aggregation causes aggregation, so you want to get rid of it.

If i were to have a blind attempt at your question, it is never a good idea to freeze thaw any protein, not even cell lysates. Thats the whole point of glycerol use, which we use in our lab at 50% (v/v) to store bead bound proteins. Thats also why antibodies and restriction enzymes are stored in glycerol since freeze thawing antibodies dissolved in water often leads to their degradation, although you only tend to store stuff in glycerol, like restriction enzymes, if the concentration of the protein is very very high. One golden trick I use is aliquoting my samples and snap freeze them in liquid nitrogen and store in -80 freezer. I'm curious as to what happens with your protein, does it degrade after a freeze thaw cycle? (I'm aware that transmembrane proteins are notorious when it comes to storage/maintenance due to their awkward hydrophobic transmembrane region so its best to speak to an x-ray crystallography expert, if there is some one like that roaming around your research lab :), since they have face problems like this quite frequently and have excellent suggestions). Are your proteins GST tagged and bound to glutathione agarose beads? or you use HIS tags and nickel beads and elute the purified protein through imidazole? i suspect it is GST in which case its probably not a good idea to snap freeze although I have (somehow) had frozen glycerol beads at -20 and they were fine after use. I have had a friend talking about protein purification and the fact that protein isolation has to be done fresh so you might have to go down that root if your current method is not ideal. Also one last question, are you purifying you protein bacterially or they get secreted into the media? I suspect if its a membrane protein you are using the cell based system since if it was E. coli you were using (as suggested above) you could have just had your protein expressed through IPTG and just washed you cell pellet in STE buffer and spun it down hard and stored the pellet at -20, in which case would save you some time in the purification process. The use of Na azide is a good idea too but I only use it for primary antibody storage which is already dissolved in the block buffer such as 5%-BSA-TBST for WB and store the whole thing at 4 oC and Na azide is there to prevent BSA and the Ab going off.

The main problem many many people have, including my self is that the majority of times, over expressed protein is insoluble or just doesn't get expressed in certain systems so you are very lucky to have a working system.

This is all I know about protein storage. I hope this helps.

Membrane Proteins

Membrane proteins are the binding proteins that mediate the conduction of ions or molecules into and out of the cell membrane. Integral, peripheral and lipid-anchored are the three typical membrane proteins.

The membrane protein is the principal constituent of the cell membrane that contributes to the plasma membrane structure. The union of membrane proteins and the phospholipid bilayer cell membrane could be temporary or permanent.

A biological layer has more than hundreds of protein at defined orientation. In this post, we will study the definition, assembling, types and functions of membrane proteins.

Content: Membrane Proteins

Definition of Membrane Proteins

Membrane protein refers to the constitutive or non-constitutive protein, which unite with the phospholipid bilayer membrane at different locations by entirely or partially spanning the plasmolemma.

Membrane sidedness” is due to the different location of various membrane proteins in the biological cell membrane, which causes asymmetry of the cell membrane. Some membrane proteins communicate with the extracellular substances, while some interact with the inner protoplasmic material.

Assembling of Membrane Proteins

We all know the proteins form through mRNA translation by the association of ribosome and transfer RNA. The ribosomes synthesize either a sequence of true signal or non-cleavable TM segment from their N-terminal.

Then, the hydrophobic segment of amino acid residues travels through the ribosomal tunnel towards the exit site. Translocon refers to the protein complex within the cell membrane that provides a ribosomal tunnel to release and insert newly synthesized proteins.

The ribosomes process the release of Tm segments relative to the encoded mRNA sequence. Signal receptor particles (SRPs) bind with the incoming polypeptide chain complex, accommodating the signal sequence in the alpha-helical conformation and halts translation.

The SR receptors interact with the ribosomes and SRPs and cause a conformational change in SRPs, allowing the transfer of the nascent ribosome chain. After the protein synthesis, the ribosome detaches from the translocon, and the released membrane proteins assume their final 3D conformation.

What is a Membrane Protein? (with pictures)

Membrane proteins are proteins that are embedded among the phospholipids that make up the bilayer structure of cell membranes. The membrane protein performs specific tasks that are essential for the proper functioning of the cell. These include transporting molecules and ions into and out of the cell, starting signal pathways and making the cell recognizable to other cells. There are two main types of membrane proteins: peripheral and integral.

Peripheral membrane proteins are embedded on one side of the cell membrane, either on the outer surface or the interior wall. Integral membrane proteins are are embedded within the cell membrane and project into the cell or the external environment. An integral membrane protein that spans the entire cell membrane, from the outer surface of the cell to the inner surface of the cell, is called a transmembrane protein.

A peripheral membrane protein that resides on the outer surface of the cell membrane interacts with molecules that are released by other cells. Through a process called cell signaling, one cell can communicate with another cell by releasing chemical messengers. The messengers are recognized by exterior peripheral membrane proteins.

The peripheral membrane proteins that are located on the interior surface of the cell membrane provide a foundation for the scaffolding of the cell. These include the cytoskeletal proteins actin and spectrin. An enzyme called protein kinase C is another interior peripheral membrane protein. It initiates signaling pathways inside the cell.

Peripheral membrane proteins do not interact with the non-polar region of the cell membrane. Instead, they are bound to the cell membrane through interactions between the polar region of the phospholipids and the polar region of the protein. Many peripheral membrane proteins bind to integral membrane proteins as well.

Integral membrane proteins transport molecules from the external environment to the interior of the cell. Some can freely transport molecules while others require energy to transport molecules. Integral membrane proteins also serve as receptors for specific molecules and as enzymes for specific metabolic pathways.

chain of an integral membrane protein can pass through the cell membrane once, or weave in and out of the cell membrane multiple times. They contain non-polar side chains, which allow them to interact with the fatty acid chains of the phospholipids. This interaction holds the integral membrane protein tightly to the cell membrane. Integral membrane proteins also have a polar region that extends into the cell or the external environment.

Guide to Protein Purification, 2nd Edition

Sue-Hwa Lin , Guido Guidotti , in Methods in Enzymology , 2009


Membrane proteins are pivotal players in biological processes. In order to understand how a membrane protein works, it is important to purify the protein to fully characterize it. Membrane proteins are difficult to purify because they are present in low levels and they require detergents to become soluble in an aqueous solution. The selection of detergents suitable for the solubilization and purification of a specific membrane protein is critical in the purification of membrane proteins. The aim of this chapter is to provide an overview for the isolation of plasma membranes, selection of detergents for solubilization of membrane proteins, and how the choice of detergents may affect membrane protein purification.

How proteins find their place in the cell

Structure of the GET insertion machine (Get1 in blue, Get2 in orange and Get3 in light blue). A representative cryo-EM image of the complex is shown in the background. Credit: McDowell and Sinning (2020)

Over a quarter of all proteins in a cell are found in the membrane, where they perform vital functions. To fulfill these roles, membrane proteins must be reliably transported from their site of production in the cell to their destination and correctly inserted into the target membrane. Researchers from the Heidelberg University Biochemistry Center (BZH) have succeeded in determining the three-dimensional structure of a molecular machine responsible for the correct placement of an important membrane protein family—the so-called "tail-anchored" membrane proteins, or TA proteins for short.

An adult human consists of an estimated 100 billion cells. Each one contains countless proteins, the architects and players in life that perform a broad range of functions. A major portion of the proteins in a cell are membrane proteins, i.e. components of the fine membranes (from the Latin membrana) that envelop every cell as well as its small organs, the organelles. Membrane proteins can form channels or pores and perform fundamental tasks such as transport of substances and signal transmission. Therefore, the correct insertion of a membrane protein is crucial for it to fulfill its biological role and, in turn, for the proper function of the cell. But what ensures that the protein ends up at the right membrane and is integrated at the right spot?

Specific signal sequences, small sections of proteins that act like "post codes", are vital for delivery to the correct location and proper insertion into the membrane. They are detected by molecular sorting machines that deliver the protein to its destination. In some proteins, the signal sequence is found at the end of the molecule, known to scientists as "tail-anchored" or TA membrane proteins. This vital membrane protein family is involved in many cellular processes, including membrane fusion and apoptosis, or programmed cell death.

BZH researchers led by Prof. Dr. Irmgard Sinning recently determined the three-dimensional structure of the molecular machine that inserts the TA proteins into the membrane of the endoplasmic reticulum (ER) - an important distribution network inside the cell that is connected to all other organelles. For their structural analyses, the BZH scientists used cryo-electron microscopy (cryo-EM), a method recognized by the Nobel Prize for Chemistry in 2017. "This type of high-resolution structural information is essential to understand the final steps of the protein insertion process into the ER membrane," explains Prof. Sinning, who directs a research group at the BZH.

The GET insertion machine is responsible for the correct insertion of TA proteins into the ER membrane. GET stands for "guided entry of tail-anchored membrane proteins". This insertion machine, which has barely changed over the course of evolution from yeast to man, consists of three protein building blocks. Two are located in the ER membrane where they form a kind of cavity (Get1 and Get2). The third one (Get3) is located outside the membrane, acting as the TA protein deliverer. All three components of the GET insertion machine are essential for the correct insertion of the TA protein into the target membrane. Get2 takes the protein from the deliverer and essentially "pushes" it towards the cavity in the interior of the membrane. The Heidelberg researchers uncovered this unexpected detail concerning the interaction between Get2 and Get3 during their analysis of the protein structure. They also showed that two copies of the insertion machine always work closely together to make the integration process more efficient. "The GET insertion machine provides the TA proteins with an energetically favorable route into the membrane," states Prof. Sinning.

"Small membrane proteins like those found in the GET insertion machine are a challenge for structural biology, so our research required innovative ideas," adds structural biologist Dr. Melanie McDowell. Only in recent years have technical improvements in cryo-EM allowed structures of increasingly smaller protein complexes to be identified in ever greater detail. Heidelberg University therefore established a cryo-EM network (HDcryoNet), making the structural analysis of small membrane protein complexes like the GET insertion machine possible. Prof. Sinning and Dr. McDowell believe that their new data provide a crucial missing puzzle piece required to complete the picture of protein transport in the cell and protein insertion into membranes.

The electrical and chemical driving force that moves ions.

The difference between the internal minus the external potential in a membrane.

An insulator or substance of very low electrical conductivity.

A device that can store electrical charge.

The space that surrounds an electric charge. For a stationary charge, the electric field E at the position where a particle of charge q is located is defined by the vector E = F/q, where F is the force exerted on the particle.

Two opposite charges of the same magnitude that are separated by a finite distance.

The transient electric current that is produced by the movement of the gating charges.

A more general term for gating currents.

An electronic device that imposes a defined potential difference across the membrane.

A plot of the voltage dependence of the gating charge.

The current that flows into and out of the plates of the capacitor during its charge or discharge.

The process of conductance reduction during maintained depolarization.

This is the human ether-a-go-go related channel, which is a potassium channel that is classified as Kv11.1.

A phosphatase that dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate to obtain phosphatidylinositol (4,5)-bisphosphate.

Part 2: Alternating Access of the Glucose Transporter

00:00:09.01 Hi. I'm Nieng Yan.
00:00:10.05 I'm a professor in the School of Medicine,
00:00:13.28 Tsinghua University, Beijing, China.
00:00:15.02 Welcome to iBiology seminar series.
00:00:17.25 In part 2,
00:00:19.13 I'd like to share with you
00:00:20.29 one major research interest in my lab.
00:00:24.03 That is, the structure elucidation
00:00:25.29 of one very fundamental physiological process
00:00:29.16 , the cellular uptake of glucose.
00:00:33.25 We all know glucose is the primary energy source
00:00:37.10 to most of the lives on Earth.
00:00:39.28 From the textbook of biochemistry
00:00:42.23 or cellular biology,
00:00:44.10 you all learned how glucose is burned
00:00:48.08 to release energy to support life.
00:00:50.14 We know through glycolysis,
00:00:52.08 one glucose molecule is split to
00:00:55.29 two pyruvate molecules,
00:00:57.06 and during this process
00:00:59.01 two ATP molecules are generated.
00:01:01.23 And in the aerobic conditions,
00:01:05.13 the pyruvate molecules
00:01:08.06 are further burned through the TCA cycle,
00:01:10.24 or the citric acid cycle,
00:01:13.02 and the electron transport chain,
00:01:15.17 to generate carbon dioxide.
00:01:20.03 And during this process.
00:01:21.20 I mean, if it's complete metabolism,
00:01:24.05 then one glucose can be used to
00:01:26.26 produce over 30 ATP molecules.
00:01:30.00 That is the energy currency for all life.
00:01:34.13 However, before the metabolism of glucose,
00:01:38.12 there is also one critical step
00:01:40.17 -- that is to take the glucose into the cell.
00:01:44.15 From part 1,
00:01:46.07 I already told you that glucose
00:01:47.28 is highly hydrophilic,
00:01:51.08 that means, they are water soluble.
00:01:52.08 However, the cell is surrounded by
00:01:56.22 the hydrophobic lipid bilayer.
00:01:57.26 So, glucose cannot enter the cell
00:02:00.02 through free diffusion.
00:02:01.13 There must be different proteins
00:02:04.24 to mediate this process.
00:02:06.18 These proteins are called glucose transporters.
00:02:10.14 So, as we see here,
00:02:14.19 glucose transporter is important,
00:02:16.16 is essential for cellular uptake of glucose.
00:02:20.07 And, throughout the years,
00:02:23.14 we have identified different types of glucose transporters,
00:02:27.01 and more glucose transporters are being identified,
00:02:31.06 but among all of those,
00:02:33.03 the most rigorously characterized ones
00:02:37.13 are called GLUTs, as shown here,
00:02:38.25 G-L-U-T,
00:02:40.26 glucose transporters.
00:02:43.18 So, in human bodies,
00:02:47.22 there is a huge family called
00:02:49.15 major facilitator superfamily,
00:02:51.07 and the GLUTs belong to this family.
00:02:52.16 Even within the GLUT family,
00:02:54.23 there are 14 different isoforms
00:02:57.01 that exhibit tissue specificity
00:02:59.28 and substrate specificity.
00:03:02.05 As summarized here, for example,
00:03:05.04 GLUT1 functions in brain and red blood cells,
00:03:08.28 and GLUT2 is for liver.
00:03:11.28 GLUT3 is also called neuronal glucose transporter,
00:03:14.27 indicating that it functions in neurons.
00:03:17.10 And GLUT4 is very famous
00:03:19.14 -- it take glucose into adipocytes and muscle cells.
00:03:24.27 So, these are the four most famous GLUTs
00:03:28.19 -- GLUT 1, 2, 3, 4.
00:03:30.13 And for the other 10 different isoforms,
00:03:32.28 unfortunately, for some of them,
00:03:35.07 their substrates remain uncharacterized.
00:03:38.24 Oh, besides, for these glucose transporters,
00:03:41.07 despite their sequence similarity with each other,
00:03:44.00 they actually may have
00:03:47.11 different binding affinities for glucose
00:03:51.06 and for other similar sugars,
00:03:54.19 and they have different turnover rates.
00:03:56.24 For example, GLUT1 can take
00:04:00.25 up to 1200 glucose per second,
00:04:04.09 but that's. yeah, that's very fast.
00:04:06.13 however, GLUT3.
00:04:08.19 for GLUT3, the number is 6000.
00:04:10.09 it's five times faster than GLUT1,
00:04:13.15 and this is amazing.
00:04:15.05 Because of their fundamental
00:04:18.02 significance in physiology,
00:04:19.28 you can imagine,
00:04:21.10 malfunction or misregulation of these proteins
00:04:24.29 are associated with various diseases.
00:04:28.17 For example, GLUT1 deficiency syndrome
00:04:31.20 is actually a rare genetic disease
00:04:34.13 manifested by early onset seizure
00:04:37.24 or retarded development.
00:04:40.10 And GLUT2, because it's associated with the liver.
00:04:43.15 so, mutations of GLUT2
00:04:46.11 are associated with
00:04:50.12 a type of disease called Fanconi-Bickel syndrome.
00:04:52.24 And more and more evidence shows that
00:04:57.02 GLUT1 and GLUT3 are overexpressed in cancer cells,
00:05:01.19 especially solid tumor cells,
00:05:04.11 because of the so-called Warburg effect.
00:05:06.01 I just told you,
00:05:08.26 without oxygen, one glucose can be
00:05:11.23 converted to pyruvate.
00:05:13.01 During this process, two ATP molecules are generated.
00:05:15.17 However, in the presence of oxygen,
00:05:18.23 that is, aromatic.
00:05:20.18 or, sorry, aerobic conditions,
00:05:22.28 about. I mean, over 30 ATP molecules
00:05:25.02 can be generated.
00:05:26.10 For solid tumors,
00:05:27.26 it's usually under hypoxic conditions.
00:05:30.23 That's. you know, that means it can only.
00:05:34.22 one glucose can only generate two ATPs.
00:05:37.10 Consequently, more glucose transporters
00:05:39.25 have to be expressed
00:05:43.14 to take more sugar to
00:05:46.08 compensate for this amounts.
00:05:47.23 And for GLUT4,
00:05:49.12 it's very famous because of
00:05:51.10 its association with type 2 diabetes mellitus
00:05:54.17 and obesity.
00:05:56.29 So, as I just mentioned,
00:05:59.03 glucose transporters belong to
00:06:01.12 the so-called major facilitator superfamily.
00:06:04.25 As a matter of fact,
00:06:06.27 they are the prototypes of this
00:06:09.10 largest secondary active transporter family.
00:06:12.22 And for members in this family,
00:06:16.00 actually, they are widespread across species,
00:06:19.04 from bacteria to human beings.
00:06:20.17 And members in this family
00:06:22.07 have a very broad spectrum of substrates,
00:06:25.22 from ions, sugars,
00:06:28.07 amino acids, or even peptides.
00:06:31.05 And in terms of transport mechanisms,
00:06:35.26 if you watched part 1 already,
00:06:39.08 actually, members in this family can be
00:06:42.02 uniporters, symporters, or antiporters.
00:06:44.15 That's in terms of the orientations of the transport.
00:06:47.00 And, as I also told you,
00:06:49.20 a general alternating access
00:06:53.18 model or mechanism
00:06:55.08 has been proposed to account
00:06:56.29 for all the secondary transporters.
00:06:58.23 Especially for MFS members,
00:07:01.05 this works very well.
00:07:02.27 And we thought that because
00:07:04.24 GLUTs are the prototypes in the understanding of this family,
00:07:07.17 so structural and biochemical characterization of GLUTs
00:07:12.17 may also shed light on the understanding
00:07:15.04 of other members of this largest family.
00:07:18.07 Okay, why is it a prototype,
00:07:20.16 especially GLUT1?
00:07:22.08 Because it was one of the
00:07:24.29 first transporters to be cloned
00:07:26.26 and characterized.
00:07:29.06 So, let me bring you to the history.
00:07:31.05 Actually, the characterization of glucose uptake
00:07:35.15 into our blood cells
00:07:38.01 can be dated back to about a century ago.
00:07:40.15 And at that time it was already discovered that
00:07:45.22 the uptake rate or the "diffusion".
00:07:47.15 at that time people didn't know it's active transport,
00:07:50.03 so they still called it diffusion.
00:07:52.25 but one PhD student found that
00:07:56.03 the diffusion coefficient is actually concentration dependent,
00:07:59.09 suggesting it was not free diffusion.
00:08:02.00 In 1948, LeFevre, in one paper,
00:08:05.24 speculated on the active transport component,
00:08:10.03 although he didn't specify
00:08:11.22 whether it was protein or something else,
00:08:13.15 but he just speculated there would be
00:08:16.15 an active transport mechanism.
00:08:19.04 And in the 1950s, Widdas,
00:08:21.00 in his very famous paper,
00:08:23.12 proposed a so-called mobile solute carrier mechanism.
00:08:26.21 As a matter of fact,
00:08:29.02 this mechanism was so famous
00:08:30.19 that all the secondary transporters in humans
00:08:35.15 are named after SLC.
00:08:38.24 So, for example, GLUT1 is actually.
00:08:41.08 the gene name for GLUT1 is SLC2A1,
00:08:45.06 but don't call it slack,
00:08:47.02 because scientists don't like that name,
00:08:49.11 so it's SLC.
00:08:51.17 And then.
00:08:53.04 and so far it's all about these components.
00:08:55.04 And in 1977, these scientists
00:08:58.18 actually were able to purify
00:09:01.14 the protein component from red blood cells
00:09:04.01 and reconstitute them into a liposome,
00:09:07.01 and they reconstituted the uptake of glucose.
00:09:10.13 So, they named this protein component
00:09:12.22 GLUT1.
00:09:14.17 And then, in 1985,
00:09:16.12 Harvey Lodish's lab cloned GLUT1,
00:09:19.20 and when the sequence was available
00:09:22.20 it was clear that this protein contains
00:09:25.16 12 transmembrane helices.
00:09:27.24 And in the 1990s,
00:09:30.26 the study efforts were shifted
00:09:33.16 to the pathophysiological investigations,
00:09:35.25 as well as structural characterizations,
00:09:38.10 because we would like to understand their structure,
00:09:41.07 to see their structure,
00:09:42.17 so as to understand
00:09:44.18 its functional mechanism and disease mechanism.
00:09:47.13 However, 30 years.
00:09:49.24 almost 30 years passed.
00:09:52.11 so what we learned from the textbook
00:09:54.01 about the structure of GLUT1 was still this,
00:09:56.26 the one published by Harvey Lodish in 1985.
00:10:00.19 This is the topological structure.
00:10:03.06 Alright, umm.
00:10:05.12 so, I started my lab in 2007
00:10:07.17 and we were very interested in the structure of GLUTs
00:10:10.14 because we thought it could help
00:10:13.00 address a lot of interesting questions,
00:10:14.25 as listed here.
00:10:16.05 Of course, the first thing is
00:10:19.01 you try to see the architecture of GLUTs,
00:10:21.18 that's the most direct, but superficial, purpose.
00:10:25.04 And with the structure
00:10:27.14 we might be able to reveal
00:10:29.11 the molecular basis underlying the substrate selectivity,
00:10:32.14 why it selects glucose,
00:10:35.22 but not, for example, maltose.
00:10:38.19 And we.
00:10:40.14 because we understand that these transporters
00:10:43.09 follow this alternating access cycle,
00:10:46.07 so we'd like to reveal the conformational changes
00:10:48.26 during the transport cycle,
00:10:50.12 to understand their functional mechanism.
00:10:54.00 And we also hope to
00:10:57.12 provide a molecular interpretation
00:10:59.10 for all these disease-related mutations.
00:11:03.26 And, for my own research,
00:11:06.27 I'm also very interested in the difference,
00:11:08.21 the mechanistic difference,
00:11:10.05 between symporters,
00:11:11.20 particularly proton symporters,
00:11:13.16 and facilitators,
00:11:15.01 but I may not have time to go into the details of this part.
00:11:17.27 And finally, because membrane proteins
00:11:21.18 are embedded in the lipid bilayer,
00:11:24.05 we would really like to understand
00:11:27.00 how they are modulated by lipids,
00:11:28.28 and there are more and more questions.
00:11:30.15 they just emerge during your research.
00:11:32.26 So, to address these questions,
00:11:34.27 we started not with a glucose transporter,
00:11:37.26 but with their relatives,
00:11:40.21 their relatives from E. coli,
00:11:42.21 which are technically easier than the human protein.
00:11:46.24 So we determined the two structures of
00:11:50.09 E. coli proton-sugar symporters,
00:11:53.02 FucP and XylE.
00:11:55.21 So, as the name indicated,
00:11:58.02 they are proton symporters,
00:11:59.12 meaning they exploit this transmembrane proton gradient
00:12:02.18 to drive the uptake of the substrates,
00:12:05.08 either L-fucose or D-xylose,
00:12:07.21 from a low concentration environment
00:12:10.20 to the high concentration interior of the cell.
00:12:13.13 In the past three years, we were very lucky.
00:12:16.27 We were finally able to determine
00:12:18.21 the crystal structures of GLUT1,
00:12:21.08 and its closely related GLUT3,
00:12:24.25 in three different conformations.
00:12:27.02 That means they adopt different states
00:12:30.04 during a transport cycle,
00:12:31.21 as shown here.
00:12:32.25 So, all the way from
00:12:35.22 outward-open, occluded, and inward-open.
00:12:37.18 When I say outward or inward,
00:12:40.08 that refers to the substrate binding site,
00:12:42.21 that is. remember, for the alternating access,
00:12:47.01 that is. the substrate binding site
00:12:48.28 can never be exposed
00:12:50.29 to both sides of the membrane,
00:12:54.09 so it's always open to one side,
00:12:55.27 the substrate comes,
00:12:57.07 and this protein undergoes conformational change
00:13:00.04 to expose the substrate
00:13:02.07 to the other side.
00:13:03.20 This is called alternating access.
00:13:05.13 So, with these three structures,
00:13:07.00 we have a relatively better understanding
00:13:08.23 of this transport cycle of GLUTs.
00:13:11.16 Alright, first thing.
00:13:13.06 To address the question of the architecture.
00:13:15.19 but, before that, I know
00:13:19.18 many people are interested in the crystallization of membrane proteins
00:13:21.28 and GLUT1 has been a target for several decades.
00:13:25.12 Why were we able to crystallize
00:13:28.19 and determine the structure
00:13:30.17 of this very intriguing protein?
00:13:31.15 In retrospect, there are three key elements
00:13:35.23 that contributed to the crystallization of GLUT1
00:13:38.26 and gave us the diffracting crystals.
00:13:41.21 First, we actually introduced point mutations.
00:13:45.01 first is to eliminate glycosylation,
00:13:47.10 which really represents major troubles
00:13:50.29 for crystallization.
00:13:52.12 And the other point mutation,
00:13:54.17 glutamate-329 to glutamine,
00:13:57.10 this one is a disease-related mutation
00:14:00.18 originally identified in GLUT4,
00:14:03.02 and it was suggested to l
00:14:05.21 ock the protein in the inward-open conformation,
00:14:08.24 which was exactly the case, as seen in our structure.
00:14:12.09 And second, on the detergent we used for crystallization
00:14:16.01 is nonyl-glucoside.
00:14:17.03 I will come back later with
00:14:19.01 why this was important.
00:14:20.04 And third, you know, for glucose transporters,
00:14:22.05 they are highly mobile,
00:14:23.14 so we would try to slow them down,
00:14:25.08 to lock them at certain conformations,
00:14:27.08 so we did all the experiments at low temperature,
00:14:29.21 at 4 degrees Celsius
00:14:31.08 -- that helped a lot.
00:14:33.06 And to cut a long story short,
00:14:35.25 one particular day my student showed me these crystals,
00:14:39.06 these tiny crystals.
00:14:40.21 I thought, probably,
00:14:43.02 they were contaminations from insect cells,
00:14:45.08 however, you know,
00:14:47.07 it doesn't hurt to send them to the synchrotron
00:14:49.29 for data collection
00:14:51.08 and we sent this single crystal
00:14:53.08 to the Shanghai synchrotron,
00:14:54.24 and several hours later
00:14:56.12 we solved the structure
00:14:58.13 that was exactly our target, GLUT1.
00:15:00.16 As shown here, this structure
00:15:04.00 exhibits a very typical MFS fold,
00:15:08.02 remember, major facilitator superfamily.
00:15:10.11 It contains 12 transmembrane helices,
00:15:13.10 with the first 6 named the
00:15:17.14 N-domain or the N-terminal domains,
00:15:19.03 shown in silver,
00:15:20.26 and the C-terminal one in blue.
00:15:23.07 And very unexpectedly,
00:15:25.01 we also see an intracellular helical domain,
00:15:28.25 we named it ICH.
00:15:30.07 Actually, this domain.
00:15:32.03 this little domain harbors
00:15:34.03 a lot of serine or threonine or lysine,
00:15:36.27 so these residues are probably
00:15:38.28 important for their post-translation modification.
00:15:42.01 Now, with this structure,
00:15:43.27 we really can provide the answer to many questions.
00:15:49.05 So, as I asked at the beginning
00:15:52.03 -- so, what is the mechanism of substrate selectivity?
00:15:55.07 For this purpose, we actually examined,
00:15:57.18 through a biochemical approach,
00:16:01.13 the sugar selectivity by GLUT1 and GLUT3,
00:16:04.09 shown here are the results for GLUT3.
00:16:06.14 As you can see, indeed,
00:16:08.13 this protein has kind of a stringent selectivity,
00:16:14.13 and one.
00:16:15.16 wherever you see these lower,
00:16:17.09 these shorter bars,
00:16:19.02 that means these sugars
00:16:21.09 can inhibit the uptake of glucose,
00:16:23.27 meaning that they can be recognized by GLUT3,
00:16:25.23 to compete for glucose binding.
00:16:28.19 And when we examined these chemicals,
00:16:31.06 very interestingly
00:16:33.09 we found one common feature,
00:16:34.23 that is, their C3 hydroxyl group
00:16:37.22 all points to one orientation,
00:16:39.23 so that means that C3 hydroxyl group is important.
00:16:43.02 That's the conclusion from biochemistry,
00:16:46.29 from our biochemical characterizations.
00:16:49.20 Then, how is one sugar molecule
00:16:52.29 recognized by the protein?
00:16:55.00 So, the answer is from the
00:16:56.24 very high resolution structure of GLUT3.
00:16:59.06 So, we determined GLUT3
00:17:02.10 in complex with its substrate, D-glucose,
00:17:04.10 at 1.5 Angstrom resolution,
00:17:08.13 and shown here is the omit electron density map.
00:17:10.19 As you can see, it's beautiful.
00:17:12.21 To our surprise, we identified.
00:17:15.11 although we just, you know,
00:17:17.20 add glucose to the protein
00:17:19.11 and we identified two different
00:17:22.05 anomeric forms of glucose,
00:17:26.01 simply by the electron density map.
00:17:27.23 As you can see, both alpha and beta glucose
00:17:31.20 are present in the structure.
00:17:33.26 I mean, I have to clarify.
00:17:35.15 so, one protein can only bind to one glucose,
00:17:39.03 but for crystallography, you know,
00:17:41.15 this is the average of many billions of molecules,
00:17:44.18 so you know some proteins bind to the alpha form,
00:17:47.16 some bind to the beta form.
00:17:49.16 And this observation,
00:17:51.07 this structural observation
00:17:53.02 actually settled down one long-term controversy,
00:17:55.13 that is, whether glucose transporters
00:17:58.19 can recognize the alpha form of glucose,
00:18:01.26 because we know the beta form is the prevailing one,
00:18:04.18 the dominant form in solution.
00:18:05.22 And this observation shows, yes,
00:18:07.28 GLUT1 or GLUT3,
00:18:09.20 they can bind and transport
00:18:12.15 both anomeric forms of D-glucose.
00:18:16.05 Alright.
00:18:17.28 Another interesting discovery is that,
00:18:19.20 as I told you,
00:18:21.04 glucose transporters have two distinct domains,
00:18:24.08 N-domain and C-domain.
00:18:26.08 However, in the structure of GLUT3
00:18:28.19 in complex with glucose,
00:18:31.17 as well as in the structure of GLUT1
00:18:34.03 in the presence of this detergent molecule NG.
00:18:37.27 what is NG?
00:18:39.04 It is actually a derivative of glucose,
00:18:41.21 so that's why NG is important for us
00:18:44.09 to capture the structure of GLUT1
00:18:46.09 -- it mimics the substrate binding.
00:18:48.13 And if you compare these two structures,
00:18:50.11 a common feature is
00:18:52.08 the C-domain provides
00:18:54.08 the primary accommodation site for glucose,
00:18:58.22 so the C-domain
00:19:01.09 is the primary substrate binding site.
00:19:04.04 Then, what does the N-domain do?
00:19:07.08 Alright.
00:19:08.14 So, before that, you know,
00:19:10.10 we tried to complete the alternating access cycle
00:19:13.09 by, you know.
00:19:16.11 in the attempt to capturing another conformation,
00:19:19.09 that is, the outward-open,
00:19:20.28 because now we have GLUT3
00:19:23.01 in complex with glucose
00:19:25.01 in the occluded conformation,
00:19:26.10 that is, the substrate is trapped
00:19:28.10 in the center of the transporter
00:19:31.20 and isolated from either side of the membrane.
00:19:34.22 And GLUT1 is open to the inside of the cell,
00:19:38.02 so it's called inward-open.
00:19:39.11 Now, we still need this outward-open conformation.
00:19:44.00 In order to capture the outward-open structure,
00:19:46.18 we really had some rational thinking.
00:19:49.04 So, people always say that
00:19:51.01 crystallization is an art,
00:19:52.12 it seems like you have to do a lot of screening,
00:19:54.12 but in this case we really did
00:19:57.10 some rational thinking.
00:19:58.09 That is, when we obtained the structure of GLUT1,
00:20:00.22 I told you NG is important, right?
00:20:04.02 So we introduced several factors,
00:20:05.22 like the mutation E329Q,
00:20:08.28 that is, to lock the inward-open conformation,
00:20:10.19 and then when we see the binding of NG to the protein,
00:20:13.28 as you can see on the tail,
00:20:16.01 the aliphatic tail of this detergent,
00:20:17.29 it actually is
00:20:20.20 lining down the intracellular vestibule,
00:20:24.18 when the sugar moeity is
00:20:27.19 specifically coordinated by the C-terminal domain.
00:20:30.06 So, along.
00:20:31.18 so, basically, the presence of this aliphatic tail
00:20:35.00 precludes the closure of these two domains
00:20:39.00 on the intracellular side,
00:20:40.22 that is, to stabilize this inward-open conformation
00:20:44.08 -- with this aliphatic tail,
00:20:46.19 it cannot close, right?
00:20:48.10 So, along this line of thinking,
00:20:50.27 we thought, if we can find a chemical,
00:20:53.07 a glucose derivative,
00:20:55.07 that has some chemical groups
00:20:57.14 on the other side,
00:20:59.08 on the upper side of the sugar ring,
00:21:01.06 probably that can preclude
00:21:04.12 the closure of the protein on the extracellular side,
00:21:07.01 that is, to capture
00:21:09.24 an outward-open conformation.
00:21:11.02 Do we have these kind of chemicals?
00:21:12.29 Yes, we have a lot of disaccharides
00:21:15.22 that are derivatives of glucose.
00:21:17.28 As shown here, we selected a few
00:21:20.15 and we examined their ability to inhibit glucose uptake.
00:21:24.22 As shown here, it turns out that
00:21:26.27 maltose was a potent inhibitor,
00:21:28.26 and when we checked the literature
00:21:30.22 this was really consistent,
00:21:32.00 because maltose was regarded.
00:21:34.06 was suggested as the exofacial inhibitor,
00:21:37.26 that means it can inhibit glucose uptake
00:21:40.16 from the extracellular side.
00:21:44.10 To cut a long story short,
00:21:45.25 in the presence of maltose
00:21:47.29 we actually crystallized the protein
00:21:50.17 using lipidic cubic phase.
00:21:52.14 It gave us two different structures.
00:21:56.01 One is almost identical to.
00:22:00.17 shown on the left, it's almost identical
00:22:01.28 to the glucose-bound GLUT3,
00:22:04.17 and is occluded from.
00:22:06.19 so, maltose is bound in the center,
00:22:08.16 occluded from either side of the membrane.
00:22:11.01 But the other crystal form
00:22:15.03 gives us this outward-open conformation,
00:22:18.22 so this was really serendipity,
00:22:21.02 I mean, we just mixed them together
00:22:22.15 and it gave us two different crystal structures.
00:22:25.12 So, I will focus on the illustration
00:22:27.25 of this outward-open conformation,
00:22:29.02 with comparison of the inward-open GLUT1
00:22:32.11 and the occluded GLUT3.
00:22:34.10 So, now we have these three conformations
00:22:36.28 I showed before.
00:22:38.06 We could generate a morph
00:22:40.03 that illustrates the whole transport process.
00:22:44.17 As you see here,
00:22:46.25 outward-open, arrival of glucose,
00:22:48.24 and it undergoes this alternating access
00:22:52.00 by the relative rotation of these two domains,
00:22:55.23 and the substrate is released
00:22:57.15 into the inside of the cell.
00:23:01.06 And, very interestingly,
00:23:02.13 remember this small domain,
00:23:05.12 shown in yellow,
00:23:06.27 is the ICH, intracellular helical domain,
00:23:09.17 and during the conformation change,
00:23:11.13 we can see it also
00:23:14.04 undergoes interdomain rearrangement.
00:23:15.26 In a way, it restrains
00:23:18.07 the N- and the C-domains
00:23:20.01 from opening too much,
00:23:21.17 so this ICH domain,
00:23:23.20 we named it the latch,
00:23:25.17 to secure this intracellular gate.
00:23:31.07 Alright.
00:23:33.03 From the movie you might think, hmm.
00:23:34.21 these two domains undergo a rigid body rotation,
00:23:36.21 but close examination of the structures
00:23:39.27 of the outward-open and occluded GLUT3
00:23:44.21 suggest, no, it's not rigid body.
00:23:46.20 Actually, we can see very sophisticated
00:23:50.06 local rearrangement of the C-domain elements.
00:23:53.27 As shown here, the one shown in cyan
00:23:57.06 is the C-domain
00:24:01.08 and the one in green is the N-domain.
00:24:02.03 Please pay attention to this TM7 motif.
00:24:06.02 You can see it undergoes a bending, a bending.
00:24:10.09 Right? This is TM7.
00:24:13.02 Not only a bending.
00:24:15.07 so, when the sidechains are shown,
00:24:17.06 you will see it actually also undergoes a rotation,
00:24:21.28 so this TM7 undergoes
00:24:24.11 very complicated local rearrangement
00:24:27.20 by bending.
00:24:29.25 the combination of bending and rotation.
00:24:32.03 So, whether this is induced by substrate binding
00:24:34.28 or this is the so-called dynamic equilibrium,
00:24:38.15 remains to be further characterized,
00:24:40.22 and our preliminary MD simulations
00:24:43.06 suggest that this is dynamic equilibrium.
00:24:47.00 even in the absence of substrate,
00:24:49.09 you can see this kind of conformational change of TM7.
00:24:53.00 Now, here's the question.
00:24:55.18 why the C-domain, shown in cyan,
00:24:58.04 is so flexible,
00:24:59.28 whereas the N-domain is just so rigid, as a stone?
00:25:03.25 And when we examine the interior of these two domains,
00:25:08.08 the answer is really clear.
00:25:09.22 So, as shown here,
00:25:12.10 the red dashes represent hydrogen bonds.
00:25:16.07 As you can see,
00:25:18.14 the interior of the N-domain is really hydrophilic,
00:25:22.28 so the high-resolution structure of GLUT3
00:25:25.18 allowed us to identify
00:25:29.02 seven water molecules within the N-domain of GLUT3,
00:25:32.12 and these water molecules, together,
00:25:34.02 interact with a set of groups of many polar residues
00:25:38.10 as a strip of hydrogen bonds,
00:25:40.01 and this stabilizes the N-domain,
00:25:45.12 so it makes it very rigid during conformation change.
00:25:48.04 In contrast, the interior of the C-domain
00:25:51.14 is highly hydrophobic,
00:25:54.12 as shown here,
00:25:55.29 so these hydrophobic residues,
00:25:57.12 they just contact each other
00:26:00.12 through Van der Waals interactions,
00:26:02.07 so they make the interior relatively greasy,
00:26:05.11 and that's easier for bending and rotation.
00:26:08.04 So, the structural analysis
00:26:10.08 really provides a good answer
00:26:12.05 to account for the distinct features
00:26:14.12 of the N-domain and the C-domain
00:26:16.04 during the alternating access cycle.
00:26:19.19 Alright.
00:26:21.06 Now, I'll.
00:26:23.01 shown here is the very simple diagram
00:26:25.03 of alternating access.
00:26:26.14 With our structures, the three structures,
00:26:27.28 we are able to
00:26:30.15 update this model with more sophisticated features.
00:26:34.24 As you can see, TM7
00:26:36.27 and also TM10,
00:26:38.18 they undergo local conformational change,
00:26:40.21 and the overall relative rotation
00:26:42.20 of the N- and the C-domains
00:26:44.11 results in the alternating exposure
00:26:48.15 of the substrate to either side of the membrane.
00:26:50.08 And, besides, please pay attention
00:26:52.19 to these yellow bars,
00:26:54.07 they are the intracellular ICH domain,
00:26:56.08 we call them the latch,
00:26:57.16 the intracellular latch.
00:26:59.05 Okay, now with the structure.
00:27:01.23 We were able to map the disease-related mutations.
00:27:06.12 Shown her is an example of the mutations
00:27:08.29 identified in patients
00:27:11.05 with the so-called GLUT-1 deficiency syndrome.
00:27:14.04 So, in total, more than 40 mutations were identified.
00:27:17.10 So, when we mapped them
00:27:19.06 onto the structure of GLUT1,
00:27:20.24 very interestingly we realized that
00:27:24.03 they clustered to three areas,
00:27:26.25 as shown here.
00:27:27.29 So, area 1 is really
00:27:31.11 involved in substrate binding
00:27:34.03 and it's easy to understand how mutations of this cluster
00:27:37.19 would affect substrate recognition or substrate binding,
00:27:40.13 hence compromising the transport activity.
00:27:44.11 And cluster 2, as shown here,
00:27:46.29 highlighted by this cyan circle.
00:27:50.22 the cyan circle.
00:27:53.09 so, basically they mapped to the interface
00:27:58.09 between ICH, N-domain, and C-domain,
00:28:01.14 and they together constitute the intracellular gate.
00:28:03.10 And, not surprisingly,
00:28:05.11 cluster 3 maps to the extracellular gate.
00:28:08.13 So, the structure really provides
00:28:11.01 a beautiful answer to
00:28:13.26 understand most of these disease-associated mutations,
00:28:16.24 so they either affect substrate binding
00:28:19.19 or the two gates,
00:28:21.03 hence affecting the alternating access cycle
00:28:24.06 of the protein.
00:28:25.18 Alright. Now, with these results,
00:28:27.12 we can address the questions
00:28:29.05 asked at the very beginning, right?
00:28:31.22 So, we know the architecture
00:28:33.16 and we provide a basis
00:28:35.20 to see the substrate selection
00:28:38.06 and we revealed three conformations of GLUT1 and GLUT3
00:28:43.08 during the transport cycle, the alternating access cycle,
00:28:48.07 and we provided some answers
00:28:49.29 to the disease-related mutations.
00:28:52.10 And, with regard to the mechanistic difference
00:28:56.10 between symporters and facilitators,
00:28:58.12 we are now doing some MD simulations
00:29:00.03 and biochemical characterizations,
00:29:02.02 and we have some tentative clues,
00:29:04.16 but this really requires further characterizations.
00:29:07.29 And now our focus has shifted to
00:29:11.02 the modulation of the transport activity
00:29:14.07 by lipids, as well as, you know,
00:29:16.09 the kinetic study of transport cycles.
00:29:18.25 And finally, we are very interested in
00:29:21.13 structure-based ligand design,
00:29:23.05 because these proteins are important drug targets.
00:29:27.06 So, with this, I would like to conclude my talk
00:29:29.15 by acknowledging the people
00:29:32.22 who made this work possible.
00:29:34.04 So, Dong, he was my postdoc
00:29:37.17 who has been the primary driver of this project,
00:29:40.17 he was leading this team of Tsinghua undergraduate students
00:29:44.00 and graduate students
00:29:45.13 to elucidate the structures
00:29:47.17 of both GLUT1 and GLUT3,
00:29:49.01 and he's now a professor in Tsinghua University.
00:29:51.23 And this work was in collaboration with many colleagues,
00:29:55.23 in Tsinghua or in the US,
00:29:57.10 as shown here.
00:29:59.21 And I'd like to thank you for watching this online seminar.

  • Part 1: Introduction to Membrane Transport Proteins

Cell biology: New molecular details about protein sorting in the cell

The targeted incorporation of proteins into the membrane is a vital process for cell maintenance these membrane proteins ensure the proper functioning of the cell's metabolism, communication with its environment, and energy supply. Protein-sorting mechanisms ensure that membrane proteins are specifically recognized among thousands of different proteins -- and are sent to the membrane, where they're needed. A team headed by Kärt Denks, a doctoral candidate in Professor Hans-Georg Koch's working group at the Institute of Biochemistry and Molecular Biology at the University of Freiburg, describes this molecular mechanism in detail in the journal Nature Microbiology, using the gut bacterium Escherichia coli. The researchers showed that the signal recognition particle (SRP), present in all living organisms, identifies correct proteins already during their synthesis.

Proteins are synthesized on ribosomes, functional units within the cell, which release proteins via a tunnel to the inner part of the cell. They are then sorted according to a pattern: Proteins to be transported contain an amino acid sequence which serves as a recognition signal for cellular sorting complexes. SRP is one of these complexes. It occurs in bacteria and in organisms with nucleated cells, and is responsible for the recognition of membrane proteins. From earlier investigations, the researchers knew that SRP recognizes membrane proteins even before they are fully synthesized. But there was debate over exactly when. At first it was assumed that the signal sequence had to have emerged completely from the ribosome protein tunnel for the membrane protein to be recognized. But subsequent work indicated that identification took place long before the signal sequence left the ribosome. The new Freiburg research confirms this.

The researchers used a technique which enabled them to examine the contacts between the ribosome and SRP right down to the level of individual amino acids -- the very building-blocks of proteins. The team showed that SRP scans the ribosome protein tunnel to find potential substrate proteins. When it recognizes a protein of the right kind, it retracts to the end of the tunnel and positions its binding pocket in order to form a stable complex with the membrane protein. Once it has done that, the SRP begins the process of moving the synthesizing ribosome to its target site at the membrane: where it binds to protein transport channels in order to anchor the protein into the membrane. If this early-recognition fails -- if for instance the contact points between the SRP and the ribosomal tunnel have been genetically modified -- membrane proteins pile up because they cannot be correctly positioned in the membrane. This leads to cell-division defects.

The research reveals a new complexity in the interaction between ribosomes and protein-sorting complexes: the ribosomal tunnel, long regarded as a passive tube, plays a key role in the coordination of processes which begin during the synthesis of proteins.

Hans-Georg Koch is the principle investigator of the German Research Foundation-sponsored research training group 2202, "Transport across and into membranes" and of the Faculty of Medicine's doctoral training group "MOTI-VATE." He is also vice-director of the Spemann Graduate School of Biology and Medicine (SGBM).


Asymmetry Edit

The lipid bilayer consists of two layers- an outer leaflet and an inner leaflet. [1] The components of bilayers are distributed unequally between the two surfaces to create asymmetry between the outer and inner surfaces. [2] This asymmetric organization is important for cell functions such as cell signaling. [3] The asymmetry of the biological membrane reflects the different functions of the two leaflets of the membrane. [4] As seen in the fluid membrane model of the phospholipid bilayer, the outer leaflet and inner leaflet of the membrane are asymmetrical in their composition. Certain proteins and lipids rest only on one surface of the membrane and not the other.

• Both the plasma membrane and internal membranes have cytosolic and exoplasmic faces • This orientation is maintained during membrane trafficking – proteins, lipids, glycoconjugates facing the lumen of the ER and Golgi get expressed on the extracellular side of the plasma membrane. In eucaryotic cells, new phospholipids are manufactured by enzymes bound to the part of the endoplasmic reticulum membrane that faces the cytosol. [5] These enzymes, which use free fatty acids as substrates, deposit all newly made phospholipids into the cytosolic half of the bilayer. To enable the membrane as a whole to grow evenly, half of the new phospholipid molecules then have to be transferred to the opposite monolayer. This transfer is catalyzed by enzymes called flippases. In the plasma membrane, flippases transfer specific phospholipids selectively, so that different types become concentrated in each monolayer. [5]

Using selective flippases is not the only way to produce asymmetry in lipid bilayers, however. In particular, a different mechanism operates for glycolipids—the lipids that show the most striking and consistent asymmetric distribution in animal cells. [5]

Lipids Edit

The biological membrane is made up of lipids with hydrophobic tails and hydrophilic heads. [6] The hydrophobic tails are hydrocarbon tails whose length and saturation is important in characterizing the cell. [7] Lipid rafts occur when lipid species and proteins aggregate in domains in the membrane. These help organize membrane components into localized areas that are involved in specific processes, such as signal transduction.

Red blood cells, or erythrocytes, have a unique lipid composition. The bilayer of red blood cells is composed of cholesterol and phospholipids in equal proportions by weight. [7] Erythrocyte membrane plays a crucial role in blood clotting. In the bilayer of red blood cells is phosphatidylserine. [8] This is usually in the cytoplasmic side of the membrane. However, it is flipped to the outer membrane to be used during blood clotting. [8]

Proteins Edit

Phospholipid bilayers contain different proteins. These membrane proteins have various functions and characteristics and catalyze different chemical reactions. Integral proteins span the membranes with different domains on either side. [6] Integral proteins hold strong association with the lipid bilayer and cannot easily become detached. [9] They will dissociate only with chemical treatment that breaks the membrane. Peripheral proteins are unlike integral proteins in that they hold weak interactions with the surface of the bilayer and can easily become dissociated from the membrane. [6] Peripheral proteins are located on only one face of a membrane and create membrane asymmetry.

Transporters Na+ Pump actively pumps Na+ out of cells and K+ in
Anchors integrins link intracellular actin filaments to extracellular matrix proteins
Receptors platelet-derived growth factor receptor binds extracellular PDGF and, as a consequence, generates intracellular signals that cause the cell to grow and divide
Enzymes adenylyl cyclase catalyzes the production of intracellular signaling molecule cyclic AMP in response to extracellular signals

Oligosaccharides Edit

Oligosaccharides are sugar containing polymers. In the membrane, they can be covalently bound to lipids to form glycolipids or covalently bound to proteins to form glycoproteins. Membranes contain sugar-containing lipid molecules known as glycolipids. In the bilayer, the sugar groups of glycolipids are exposed at the cell surface, where they can form hydrogen bonds. [9] Glycolipids provide the most extreme example of asymmetry in the lipid bilayer. [10] Glycolipids perform a vast number of functions in the biological membrane that are mainly communicative, including cell recognition and cell-cell adhesion. Glycoproteins are integral proteins. [2] They play an important role in the immune response and protection. [11]

The phospholipid bilayer is formed due to the aggregation of membrane lipids in aqueous solutions. [4] Aggregation is caused by the hydrophobic effect, where hydrophobic ends come into contact with each other and are sequestered away from water. [6] This arrangement maximises hydrogen bonding between hydrophilic heads and water while minimising unfavorable contact between hydrophobic tails and water. [10] The increase in available hydrogen bonding increases the entropy of the system, creating a spontaneous process.

Biological molecules are amphiphilic or amphipathic, i.e. are simultaneously hydrophobic and hydrophilic. [6] The phospholipid bilayer contains charged hydrophilic headgroups, which interact with polar water. The layers also contain hydrophobic tails, which meet with the hydrophobic tails of the complementary layer. The hydrophobic tails are usually fatty acids that differ in lengths. [10] The interactions of lipids, especially the hydrophobic tails, determine the lipid bilayer physical properties such as fluidity.

Membranes in cells typically define enclosed spaces or compartments in which cells may maintain a chemical or biochemical environment that differs from the outside. For example, the membrane around peroxisomes shields the rest of the cell from peroxides, chemicals that can be toxic to the cell, and the cell membrane separates a cell from its surrounding medium. Peroxisomes are one form of vacuole found in the cell that contain by-products of chemical reactions within the cell. Most organelles are defined by such membranes, and are called "membrane-bound" organelles.

Selective permeability Edit

Probably the most important feature of a biomembrane is that it is a selectively permeable structure. This means that the size, charge, and other chemical properties of the atoms and molecules attempting to cross it will determine whether they succeed in doing so. Selective permeability is essential for effective separation of a cell or organelle from its surroundings. Biological membranes also have certain mechanical or elastic properties that allow them to change shape and move as required.

Generally, small hydrophobic molecules can readily cross phospholipid bilayers by simple diffusion. [12]

Particles that are required for cellular function but are unable to diffuse freely across a membrane enter through a membrane transport protein or are taken in by means of endocytosis, where the membrane allows for a vacuole to join onto it and push its contents into the cell. Many types of specialized plasma membranes can separate cell from external environment: apical, basolateral, presynaptic and postsynaptic ones, membranes of flagella, cilia, microvillus, filopodia and lamellipodia, the sarcolemma of muscle cells, as well as specialized myelin and dendritic spine membranes of neurons. Plasma membranes can also form different types of "supramembrane" structures such as caveolae, postsynaptic density, podosome, invadopodium, desmosome, hemidesmosome, focal adhesion, and cell junctions. These types of membranes differ in lipid and protein composition.

Distinct types of membranes also create intracellular organelles: endosome smooth and rough endoplasmic reticulum sarcoplasmic reticulum Golgi apparatus lysosome mitochondrion (inner and outer membranes) nucleus (inner and outer membranes) peroxisome vacuole cytoplasmic granules cell vesicles (phagosome, autophagosome, clathrin-coated vesicles, COPI-coated and COPII-coated vesicles) and secretory vesicles (including synaptosome, acrosomes, melanosomes, and chromaffin granules). Different types of biological membranes have diverse lipid and protein compositions. The content of membranes defines their physical and biological properties. Some components of membranes play a key role in medicine, such as the efflux pumps that pump drugs out of a cell.

Fluidity Edit

The hydrophobic core of the phospholipid bilayer is constantly in motion because of rotations around the bonds of lipid tails. [13] Hydrophobic tails of a bilayer bend and lock together. However, because of hydrogen bonding with water, the hydrophilic head groups exhibit less movement as their rotation and mobility are constrained. [13] This results in increasing viscosity of the lipid bilayer closer to the hydrophilic heads. [6]

Below a transition temperature, a lipid bilayer loses fluidity when the highly mobile lipids exhibits less movement becoming a gel-like solid. [14] The transition temperature depends on such components of the lipid bilayer as the hydrocarbon chain length and the saturation of its fatty acids. Temperature-dependence fluidity constitutes an important physiological attribute for bacteria and cold-blooded organisms. These organisms maintain a constant fluidity by modifying membrane lipid fatty acid composition in accordance with differing temperatures. [6]

In animal cells, membrane fluidity is modulated by the inclusion of the sterol cholesterol. This molecule is present in especially large amounts in the plasma membrane, where it constitutes approximately 20% of the lipids in the membrane by weight. Because cholesterol molecules are short and rigid, they fill the spaces between neighboring phospholipid molecules left by the kinks in their unsaturated hydrocarbon tails. In this way, cholesterol tends to stiffen the bilayer, making it more rigid and less permeable. [5]

For all cells, membrane fluidity is important for many reasons. It enables membrane proteins to diffuse rapidly in the plane of the bilayer and to interact with one another, as is crucial, for example, in cell signaling. It permits membrane lipids and proteins to diffuse from sites where they are inserted into the bilayer after their synthesis to other regions of the cell. It allows membranes to fuse with one another and mix their molecules, and it ensures that membrane molecules are distributed evenly between daughter cells when a cell divides. If biological membranes were not fluid, it is hard to imagine how cells could live, grow, and reproduce. [5]

Conclusion and perspectives

The use of nanodiscs is rapidly spreading, as facilitated by ready access to the genes encoding membrane scaffold proteins from AddGene and purified proteins from Sigma or BioNanoCon. In addition to the established applications of nanodisc technology, some recent developments have suggested new ideas, such as the generation of therapeutic antibodies against antigens incorporated into nanodiscs 68 and various in vivo uses of nanodiscs, including drug delivery 69 , imaging 70 and vaccine development. An exciting application is the ability to generate soluble MP libraries that faithfully represent a starting MP composition, with individual nanodiscs carrying a specific target 5 . By allowing high-throughput screening of MPs, nanodiscs can potentially facilitate the discovery of new approaches to therapeutic intervention.