September 2017

Pumps and gradients

Gradients


As we said, the membrane is impermeable to most dissolved solutes. Gases can pass freely through the membrane, so oxygen and CO2 can be traded as necessary. But things dissolved in the water that surrounds the cell and is inside the cell generally are hydrophilic. As such, they cannot pass through the membrane directly.
For this reason, there are transport proteins that are responsible for moving all of the small molecules, ions and even fairly large molecules across the membrane. We will talk a little bit later about the protein that allows water to move across the membrane. For right now we will talk about transport in terms of several overlapping concepts shown in the diagram.


transportImage from www.physiologyweb.com

Passive diffusion


all forms of passive diffusion are "down gradient." That is to say, one side of the membrane has more of the solute while the other side has less. Given any pathway through which the solute can move it will tend to distribute more evenly. I can divide this further into simple diffusion for molecules that can diffuse again across the membrane directly, passive diffusion for molecules that pass through a channel protein (which is just what it sounds like, a protein that forms a channel through the membrane), and facilitated diffusion, where the solute is carried by a protein from one side of the membrane to the other, sort of like a shuttle. The important thing to remember is that all three of these are diffusion down gradient and require no input energy. No work needs to be done for molecules to diffuse. In fact as we will see later, a gradient can be used to do work.

Active transport.


All forms of active transport require the expenditure of energy at some point in the overall pathway and result in even greater disparities in concentration between the two sides of the membrane. That is, something moves from an area where there is a low concentration to an area where there is already a high concentration of that solute.
Active transport can be separated further into "pumps," which directly expend energy in a cycle to move solutes from one side of the membrane to the other, and "co-transport." Co-transport uses a gradient that was established by a pump or exists for some other reason to drive transport of some other molecule up its own gradient. This is one of those things that is hard to explain in writing. However the diagram below does a pretty good job of showing it.
Symport is when the molecule that is moving down gradient is moving in the same direction as the molecule you want to move up gradient.
SucroseTransportimage from your textbook, Campbell Biology ninth edition

In the diagram, we see that ATP is expended to pump protons to the external side of the cell membrane.Those protons, given a pathway, would diffuse down their gradient back into the cell. The trick here is to use a pathway (that is to say, a protein in the membrane) that will only allow protons to diffuse down gradient if a sucrose molecule accompanies it.
You can think of it sort of as a revolving door that will allow protons to enter but will only revolve if sucrose is there also.
The other related concept is the "antiporter." you can think of this as a revolving door where the two players have to enter opposite sides in order for the door to turn. In either case one molecule or ion diffuses down its gradient (passive diffusion) while the other molecule moves up its gradient (active transport).

Ion pumps and the electrochemical gradient


In order to carry out co-transport, one needs to have a gradient established of one or more ions. This is generally achieved by proteins that we call "pumps." While there are many that are crucial to cell function, one has an outsized role in establishing what we call the "electrochemical gradient."That protein is the sodium-potassium pump. Sometimes you will hear it called the sodium-potassium antiporter ( which makes sense because sodium and potassium ions are moving in opposite directions – but is misleading since both will move up gradient against the direction favored by diffusion.) Another name you may hear is the sodium/potassium ATPase.
Here is a moderately
amusing GIF based on Drake's music video of a couple years ago.
Here is the gist of it: the pump can bind two potassium ions when it is open to the outside, but cannot find sodium ions at all. When open to the inside it will no longer bind potassium ions, but can bind three sodium ions. ATP is hydrolyzed to force the switch between the two states (open to the inside versus open to the outside). The net result of this is that as it goes through a cycle two potassium ions are pumped into the cell and three sodium ions are pumped out. This results in a gradient of potassium (high on the inside, low on the outside) and sodium ions (high on the outside, low on the inside). This is the "chemical" part of the gradient. Because two positive ions are pumped in for every three that are pumped out, there is also an electrical component to the gradient. The inside of the cell ends up being negatively charged overall to a level of about -70 mV.

I would like you to read David G’s really nice blog on the pump. I think you will find some of the numbers, frankly, shocking. Also, understanding the way this works will help you with other proteins we consider.
For example, some may be interested in proton pumps in the cells of the stomach that make the stomach acidic...it works like this protein, as David explains.

Proceeding from specific to general:

The Pump


  • This protein has binding sites for ions. Below is an image of two ions bound. In this case, the protein crystallographers have used a trick and bound Rubidium+ to the sites. But, it works similarly. What would bind a positive ion? Well, you should hold it between negative ions, like deprotonated acid side chains. What are the acid side chains in amino acids?: Aspartic Acid (D) and Glutamic Acid (E). Look at the image below:

  • IonBindinginPump
  • D804, E327 and E779 (the numbers correspond to the residue number in the amino acid sequence, or primary structure) all come together to make a really nice pocket that binds the ions (greenish). That makes sense. There are others I didn’t label. All three residues that bind the cation are on different helices. If you slide those helices relative to each other, the components of the binding site move closer or farther away from each other. Now, check out the ionic radii of Na+ and K+ at the wikipedia page. K+ is 30% bigger than Na+ (Rb+ is only slightly larger than K+). So, by sliding the helices, I can change the sized of the pocket and whether K+ or Na+ is favored.
  • ATP hydrolysis transfers a phosphate to a serene on the pump (Serine 33 on our pump), causing the helices to shift. This opens the pump to the outside and changes the shape of the binding pocket so that Na+ cannot really be held tightly anymore and K+ fits in well.
  • The Binding of K+ changes the shape a little more. This favors hydrolysis of the phosphate from the serine, shifts the helices back to be open inside the cell and makes the pocket too small for K+, but allows Na+ back in. Of course, I didn’t explain exactly, but the three Na+ sites only reconstruct 2 K+ sites once the helices move.

This type of pump could be modified to pump other cations.


  • As Goodsell points out, this same mechanism can be used to pump protons. You just have to change the size of the binding pocket. That probably just involves some shifting of the positions of the acidic residues.

Gradients are useful


  • Once you establish a gradient, you can use it to do stuff. The pump establishes an electrochemical gradient. Every cell at “resting potential” has more K+ on the inside and more Na+ on the outside. The inside is also more negative. Maybe I could use that gradient to do something.
  • For example, suppose I had a protein that would allow Na+ to come back in...but it would only do that if there was also a glucose molecule bound. I could use the gradient to transport glucose into the cell.
  • Suppose I had a channel that I opened in response to some signal...maybe something binding to the channel, and that channel would allow sodium to pass through it. All the sodium ions would start racing into the cell, down their chemical gradient. But that would also make the inside positively charged...that would be a dramatic effect. It get’s even more complicated, but that’s the start of an action potential in nerve cells.
  • Or...what if I stacked a whole bunch of cells on top of one another and made a little battery of cells, each with a small voltage across the membrane...then released all of them at once. That would be really interesting.
  • Phosphorylation and interactions with other molecules change the shape of proteins

  • A more general mechanism exists by which any protein could have a set of possible shapes, each favored under different conditions. Imagine a protein that binds to microtubules, for example. Maybe you could come up with a series of shapes that change depending on whether ATP is bound, or ADP is bound, or the protein is bound to tubulin or not... Could you imagine a series of steps like: Bind ATP and Release from tubulin; hydrolyze ATP and move forward; bind again, release ADP....what would that look like?

Cells intro

  • All living things are made of cells

  • Why? Because I said so. Well, not because I said so. This is something called the “cell theory of life” and it builds into our definition of life the requirement for cells. There are a lot of ways in which this makes sense. However, there are many biological entities, most notably, viruses, which are not cellular and at least fulfill many of the other requirements for being alive.
  • There is a pretty good interactive site here.
  • Components of a cell

  • We looked at diagrams of “generic” animal and plant cells (by generic, I really mean cells that don’t look like any real cells.
  • I want you to know the names and general descriptions of the major organelles.

  1. Nucleus: large structure that houses the chromosomes (DNA). All RNA for the cell is made there, using the DNA as a template. It is encased by a double membrane. That is, two complete lipid bilayers. There are pores, or holes, for the transport of stuff out.
  2. Nucleolus: dense structure in the nucleus. It is the site of ribosome assembly. Ribosomes are then transported to the cytoplasm through the pores. Some scary numbers: a single mammalian cell may have 10,000,000 ribosomes and about 10,000,000,000 protein molecules (numbers form British Society for Cell Biology).
  3. Rough endoplasmic reticulum: Membrane stack near the nucleus. Site of synthesis of proteins that either end up in membranes or are secreted out of the cell (secreted merely means transported out of the cell). They are “rough” because they are dotted with ribosomes.
  4. Smooth endoplasmic reticulum: Lacks ribosomes. Has many varied functions depending on cells. Is the site of phospholipid synthesis, steroid synthesis and used to manage calcium ion concentrations in cells that really use a lot of that.
  5. Golgi apparatus: Stacks of membranes that are between the rough ER and the plasma membrane. Proteins are transported from the ER to the Golgi in spheres of membrane called “vesicles.” In the Golgi, proteins are processed (usually meaning sugar molecules are added to specific amino acid residues).
  6. Plasma membrane or cell membrane: the lipid bilayer that keeps the outside out and the inside in. It also has many proteins in it, which may function, for example, as channels allowing transport of nutrients, or allow cells to grab other surfaces or detect the presence of various molecules.
  7. Lysosomes: Spherical membranes that derive from the plasma membrane. Imagine them as pinching off from the membrane and taking in with the membrane any proteins in the area that pinches off. They are sites for “recycling” components of proteins.
  8. All together, numbers 3-6 (actually starting at the nuclear envelope) make up sort of one continuous super-organelle comprising a system of membranes. If I were to make radioactive lipids an put them in the cell, so I could detect the radiation where ever it was, I would find the “hot” show up first in the nuclear envelope, and then later would be detected in each of the other places as time passed.
  9. Mitochondria: these are the sites in which the high energy molecules from your food are broken down to low energy products (water and CO2) and the released free energy is captured in other high-energy molecules such as ATP, used to help run many reactions in your body. Mitochondria have their own chromosome (circular, like bacteria), their own ribosomes and their own tRNA. The function of all these components resembles that of bacteria. It is thought that ribosomes derived evolutionarily from bacterial cells that became part of a larger cell.
  10. Cytoskeleton: worst representation ever in the diagrams. We will look more at this later. It is a network of fibers made of the proteins actin (microfilaments), tubulin (microtubules) and Keratin (intermediate filaments…actually, there are many forms of each of these proteins). Microfilaments and microtubules are constantly changing. Microfilaments are usually involved when cells are migrating, are found more at the edges of the cell and at points where cells are attached to surfaces. Microtubules all grow out of centrioles or similar structures (generically known as “microtubule organizing centers” or MTOC). In a generic cell, that usually is the centriole, near the nucleus. However, the tail of sperm and many other things that stick out of cells and allow them to move (wag like the tail of sperm…called either “flagella” or “cilia” are made of microtubules and they each require their own set of centriole-like structures). Intermediate filaments are more permanent and are used to link cells together in very strong tissues (like skin). You only find them in cells that aren’t going any where anytime soon.

Unique to plants:



  1. Cell wall: cellulose-based structure that provides rigid “box” around cell.
  2. Vacuole: large, fluid-filled region of the cell, surrounded by membrane. Used to maintain pressure in the cell, keeping the cell pressed out against the cell wall.
  3. Chloroplast: site of photosynthesis. Like the mitochondria, these have their own DNA, ribosomes and tRNA. They also appear to have been bacterial in origin…long ago.
  4. “Higher” plants (all the plants you know well…except maybe kelp, don’t have true centrioles. This is really a distinction without a difference. They have MTOCs, but lack the clear cylindrical tubes that we call centrioles.
  5. Plasmodesmata: holes in the cell wall that allow direct diffusion of medium-sized things between cells (we will see that animal cells have smaller pores between them called "gap junctions").
  6. Peroxosome: similar role to lysosome.

Below I’m including some real micrographs of what the components of cells look like. I can explain the technique in great detail later, if you like. But, for now, just know that we have tools that allow us to make any structure we want “fluoresce” or “glow” in a special microscope. These are not the best images. But you get the idea.
CellPhotos

Science Meme

Ran across this meme and thought might be both funny and educational.

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DNA/RNA 1

Lambda_repressor_1LMB
Wikipedia wikimedia commons image of a Short stretch of DNA bound by a protein called “Lambda Repressor.” DNA bases are shown in “stick model” form, sugar-phosphate backbone as red or blue pipes. Protein is shown in “ribbon” form, backbone only (no sidechains).
This is one of the ways that genes are turned “off” and “on.” In this case, it’s for a gene found in a virus that infects bacteria. But, many of the same principles will apply all over.
The Double Helix
This is a really beautiful structure. Unfortunately, the beauty lies in just how much it explained when it was first proposed, and is therefore, perhaps, hard for you to see. To me, it is astonishing. I’ll run down a few features.
Basic Structure (image also from Wikimedia Commons):
514px-DNA_chemical_structure
And a more detailed view:
340px-DNA_Structure+Key+Labelled.pn_NoBB
  1. There are two strands. Each is defined by a polymer of sugar-phosphate moieties (yes, that’s a word similar in meaning to “functional group”). Each subunit is linked from the number 3 carbon of one deoxyribose, to a phosphate, then to the number 5 carbon of the next deoxyribose. Instead of calling them 3 and 5, we call them 3’ (pronounced 3-prime) and 5’. The “prime” tells you that the number refers to the carbons in the sugar, as opposed to a carbon in the rings of the “base.”
  2. Thus, each strand has a chemical polarity. What I mean is that each strand has a 5’ end and a 3’ end, just as protein has an amino end and a carboxyl end. That, it turns out, is not a coincidence.
  3. The strands are aligned “anti-parallel.” The 5’ end of one strand of the double helix is aligned with the 3’ end of the other.
  4. The information is encoded by the “bases,” which protrude from the polymer backbone. This is really one of the key points: DNA is a code. The sequence of bases stands for something else. The main thing a DNA sequence stands for is the sequence of amino acids in a protein. A good first definition of “Gene” is: a stretch of DNA that encodes a protein.
  5. The information takes the form of hydrogen bonds. This is more profound than it seems: information is energy. Adenine has a much greater ability to bond to Thymine than to Cytosine; Guanine bonds much more tightly cytosine. Thus, we have A-T pairs and G-C pairs. Each base “complements” the one it pairs with in terms of hydrogen bonds it can make. We use the term “complementarity” to describe this.
  6. Now, for some interesting stuff: DNA is redundant. I only have to see one strand to know what the sequence of the other is. Each stand has all the information needed to specify its opposite strand. When there is damage to one strand, the other strand tells you how to fix it. To replicate, each strand directs the synthesis of its mate.
  7. The information is in the hydrogen bonds...and it’s a code that specifies the primary sequence of proteins (among other things). Yes, I am aware that I too am being redundant. It’s important. The DNA and the peptide sequence are “co-linear.” The 5’ end of a gene corresponds to the amino end of the peptide chain. The 3’ end of the gene corresponds to the carboxyl end of the protein. Each group of three bases in a gene is a “codon,” specifying what amino acid goes in the corresponding place of the protein...Obviously, some fascinating machine and lots of regulation must go into that. I could go on and on...but I won’t just yet.

RNA

  • ATP
  • This is ATP (adenosine triphosphate). You’ve heard of it in terms of an “energy source” for your body. That’s true, as far as it goes. However, It is also one of the four bases that are the building blocks of RNA. The difference between this and dATP (“d” for “deoxy”) is the presence of that oxygen on the 2’ carbon. (Note, when I draw it, I usually have the protons removed from the phosphates. It’s an acid and will deprotonate under most conditions in the cell. this diagram I lifted happens to have the phosphates protonated).
  • Here are three important differences between RNA and DNA:
  • There is the extra oxygen.
  • RNA is almost always single stranded, but that strand can fold back to form stretches of helix. Remember that if you fold a strand back, the arrangement is antiparallel, which is the way nucleic acids align when pairing.
  • The rules for base-paring are the same…except that a different pyrimidine, “Uracil” or “U” stands in for Thymine. It’s pairing is no different. It just lacks a methyl group on the pyrimidine ring.
  • All RNAs in the cell are made by making a copy of DNA. That is, there is DNA in the cell that corresponds to all the types of RNA we have. This will expand our definition of “Gene” a little.
  • There are three main types of RNA in the cell (that’s a lie…there are more). But, the three main types for now are:
  • mRNA: the message form of the code. The mRNA will look just like the coding strand of DNA, except it will have uracil instead of thymine (and have the 2’ oxygen). The machine that translates the code into a protein sequence does not read the DNA directly. mRNA is sort of a "buffer" copy of the information. DNA, in this analogy, is the permanent "archival" copy of the gene.
  • rRNA: The structure of the machine that reads the code and synthesizes protein. This machine is called the “ribosome” and actually includes proteins as well. It is the RNA that is the major player.
  • tRNA: transfer RNA is the “adapter” molecule. It is an RNA and therefore can “read” the code using base-pair rules. But, at one end of it, there is a specific amino acid. So, the tRNA that reads the codon UGG in messenger RNA will have a phenylalanine attached to the end of it, because UGG is a “codon” for phenylalanine (phe, or simply “F”).
  • Below is the structure of the Phenylalanine tRNA.
  • 220px-TRNA-Phe_yeast_1ehz
  • Notice that it has a detailed structure, not unlike a protein. In this case, the “secondary structure” is mediated by base-pairing. Where protein has helices and sheets, RNA has “stems” and “loops.” I’m pretty sure you can see in the cartoon to the lower right of the structure what a stem and loop is. The stems are regions of base pairing and loops have free, non-base-paired bases. That blue loop at the bottom is the “anticodon loop.” In the gray section it has the sequence 5’ CCA, so that it reads the 5’UGG codon. It may seem more natural to write the anticodon as ACC to show that it pairs with UGG. However, I will almost always write 5’ to 3’.
  • It also has tertiary structure, folding up in the “L” shape you see. That is also mediated by base pairing and other interactions.
  • You know it’s funny, you would think looking at this structure that we would have guessed that RNA like this could have protein-like functions. I mean…it has structure and functional groups (the most important ones, I"ve told you). Well, nobody guessed it (except Francis Crick). But, it turns out to be true. I’ll talk more about that later.
  • The wikipedia entry has some nice, simple pictures of translation--the making of proteins using the mRNA sequence as a guide. We’ll cover that later. But, if you want to take a look.