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Biology (Sci)
BIOL 200
Richard Roy

Krishnaa Siva Protein: Structure, Function and Separation -Mouse experiment with feeding behaviors- Goody related neurons in mice, which has a role in feeding behaviors - Heterologous: coming from different organisms or context -Channelredopsin is like the transgene that is introduced into neuron/cell type, they are introduced using promoters that drive expression in those particular neurons -Mouse is expressing channelredopsin in particular neurons, channel redopsin channels opens when pulses of shiny blue light is shined at it, allows ion flow and activation of depolarization of the neuron related to feeding behavior -how to get the genes in: use tenuated viruses that will express genes when they infect particular genes, introducing vectors early in development through microinjection (engineered so that it is not performing the viralytic cycle or infect nastily but can reproduce with every cell division) -how to induce particular behavior: isolating neurons, identifying neurons, isolating how we can activate them using ChR -Promoter is what determines where the gene is expressed (ex. neurons), like it can be tissue specific context specific, promoter confers the specifity (what cells express a given gene at a given time), drives the expression of the genes in a restricted way Some proteins have very interesting properties -green fluorescent proteins can be used to see when cells expresses a gene, its a reporter, instead of expressing the gene, GFP will be expressed when it is substituted for it, "that gene is expressed at that time at a specific cell" -NEURON:... expressing very strong the GFP, once translated from mRNA it folds up into some functional protein that gives of light when you shine a particular wavelength light, proteins can fold up into various structures and do various applications within cell(function) second picture: by looking at GFP structure, interesting shape in 3D, it looks like a barrel, in the middle of the barrel it binds oxygen, barel structure and O2 binding is very important for its ability to emit fluorescence -it allows to engineer protein based on that structure, instead of just green we have red, yellow, cyan florescent proteins, that have minorly changed GFP so that it emits at different wavelengths, sometimes just used as an idea to build on and create more proteins Structural characterisation links primary sequence to protein topology -Structure and function are not always revealed from primary sequence (amino acid sequence info)- impossible to predict how the "thing" is going to fold up in 3D Shape -By determining protein structure we can better understand how proteins carry out their respective functions -Pictures: 7 membrane pass receptor like molecules , when you look at primary sequence on top , there is no evidence that it will fold up into these elaborate structures at the bottom, we know there are hydrophobic residues cycling within amino acid (from primary sequence), did not know much till we cloned, characterize -primary sequence on top: Amino acid chains will go in and out between cytoplasm and external region through the membrane, in the membrane there is hydrophobic residues so it is ok there and then we end up structure on bottom Crystallography and X-ray diffraction have been useful to identify protein structure • High concentrations (first biologists will produce large quantities of protein that could be in bacteria or in some other vector then you play with conditions and get proteins to form stable crystal latices that you can use analyze with x-ray diffraction, this can take 1000s of different attempts with different conditions) of purified protein can organise to form crystal lattices -Crystals ensure that proteins can pack to gether in a low energy form and form latices • These crystals can be bombarded with high energy beams (ie..X-rays beam) which are scattered according to the atomic arrangement of the atoms within the crystal. - the beam will pass through the protein crystal and most of the energy is going to go right through, once in a while we have deflections, around where we are blasting the crystals, we will have sensitive detectors, every time you get a particle/crystal that bounces on the detector screen you will mark it) • The scatter pattern is detected by radioactive detectors and the data is analysed by complex programs to provide an prediction of electron density • “inferring the shape of a rock/protein by examining its ripple pattern/diffraction patterns that forms on a smooth surface of a pollen for example, …”We can go back and determine what the atomic composition of the protein looks like in that crystal latice Electron density maps provide a skeleton upon which one can build a model • By recording the electron density maps structural biologists can begin to build models by filling in the observed density with amino acids that correspond to such shapes -electron density based on the diffraction pattern -fit amino acid to the various electron density maps we established and build a space fitting model, of how the amino acids come together to give the electron density map we detected through diffraction pattern -that allows us to put together an amino acid chain, taking into consideration how everything folds to give rise to final structure (starts from ripple pattern) • Amino acids are linked into ball and stick models to complete the chain and then secondary and tertiary structures along with individual interactions (hydrogen bonding, van der Waals interactions…) between residues can be highlighted Proteins can be catalytic-Enzymes -structures of protein dictate their function and behavior, you change structure, affecting their function • Enzymes reduce the energy required to carry out a given reaction to take place within a cell -proteins typical of enzymes, it has to recognize a substrate, interphase on protein that is for the substrate(s) interaction= substrate binding site -mostly, proteins require an independent catalytic site, which means there's amino acids that will be critical in carrying out specific catalytic event that will take place that is typical of that enzyme -sometimes catalytic site near substrate binding site, but b/c of the way it can fold up in 3D shape, it can be on the other end, may end up folding over and being next to the substrate • Often enzymes enhance substrate interactions such that reactions are favoured • Critical residues in the active site may be involved in ternary complex formation and drive reactions forward Quantification of catalytic efficiency can reflect specific properties of a protein -Not all substrates are used at the same right, you can characterize enzymes by playing with substrates -some substrate will fit easily into a substrate binding site, and will be quickly catalyzed into a product, other substrates will be less optimal, they will still fit in to same binding site and catalyzed to form product, these become characteristics of that protein -the more substrate you add to limited amount of enzyme/protein, will give rise to a certain amount of conversion to its final product until you arrive at a "plateau"=Vmax, if you add another enzyme, it will go twice as fast, or four times as fast as shown in this graph but you still arrive at a Vmax, until you are saturated, you are at a plateau, ur not limiting for substrate (when you start limiting for substrate you can see how efficient this enzyme can be for the substrate) ex. arm takes red smarties, sometimes takes other colors, when my arm is working as fast it can , I got all the red smarties i want, it can not go any faster or do any better, that's a characteristic of me -Vmax does not tell anything about the enzyme (just tell how fast enzyme can go), b/c at Vmax no matter how much you give it, it is always the same, but the 1/2 max tells something about the enzyme -1/2 max says something about the enzyme, its ability to use substrate to catalyze to give a certain product -whether there is 1x enzyme or 4x enzyme (like from the 2 lines on picture) the 1/2 max does not change, it falls at the same concentration of substrate -1/2 max- Km= Michaelis Constant (characteristic of protein, does not change with levels, substrates, or the amount of enzymse)=Absolute value, place it works at 50% of maximum ----this is for one type of substrate, when theres another one, complicated -when you put in pink ones, and you end up picking of pink by mistake instead of red, Km will change -Km helps know how much substrate you should put for the enzyme to work efficiently- if its too low the enzyme will not function properly, we will not get a good rate of formation, ex. Polynucleotide kinase, enzyme need to work well to transfer the phospharayse from ATP Proteins can bind other molecules • Proteins may interact specifically with other molecules-some small some large • Ligands ≈ Binding entities (that will associate with proteins) -it bind to other lipids, other proteins or small peptide • Some common ligands include: Growth factor -> receptor (small polypeptides that will react with more complex receptors, will activate complex series of events downstream involved in signal transduction Steroid hormone -> receptor (small lipids based on cholestrol backbone, will bind to complex receptors, & initiate a whole transcription of bunch of genes) Cytokines -> receptor (small molecules bind to receptors) ligands can be small interactions between proteins -it can be enzyme-substrate, 2 proteins coming to form a complex • Ligand binding can change protein conformation considerably: Allosteric switches (AMP Activated (Protein) Kinase will phosphorylate a number of different targets, when it is bound to AMP(indicates energy level in cells), phosphorylate much higher than it would normally would without AMP) - allosterically activated on an independent site of the protein kinase that will enhance its activity • Strength of a protein:protein interaction can be expressed as • Kd- Dissociation Constant ---its not [Protein A] [Ligand/Protein B] K d [Protein A-Ligand/Protein B] a minus i think, it just means bound protein -the bottom can be the association complex Protein=ligand, small molecule... -when Kd is very very small 10^-9, 10^-12 this suggests that the proteins interact very strongly, b/c the amount of free protein over the amount of bound protein is very small, it likes to form a complex, it tells us about the affinity it has for one another -enzyme and substrate binding is ligand binding too Calcium, Calmodulin and conformation… -Calmodulins has EF hand domains (EF1, EF2, EF3, EF4), it is a coofactor • Many proteins require Ca for optimal function. Often this requires a Ca binding protein called calmodulin. 2+ • Calmodulin radically changes its conformation when bound to Ca • In the Ca -bound state calmodulin can recognise specific regions of proteins to which it binds-thereby altering protein function in a Ca -dependent manner. Proteins can act as switches -proteins that act as triggers ((small)GTPases(Gproteins) in general, Protein Kinases) -Small GTPase bind to GTP (glonning) nucleotides, when they are bound to GTP these proteins are on their onn state, there is an intrinsic GTPase activity(they will always hydrolyze a little), there is GAP activitity, that will enhance the intrinsic GTP hyrolysis within the proteins themselves (in that sense it turns it on, ), interaction with the GAP will enhance that activity, and will hydrolyze that phosphate to give rise to GDP, when protein is bound to GDP it is off, in this GAP helps turn it off, GAP is specific for GTP binding proteins that act key regulators of the trigger within the cell -when you want to turn it on, you want to kick the GDP out of the binding site and replace it GTP to put it into its active formation and thi
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