Cell Biology Exam 1 Notes
Cell Biology Exam 1 Notes BIOL 3030-001
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Cell Biology Exam I Chapter Three: Protein Structure and Function Part I: Hierarchical Structure of Proteins Many different types of proteins that have evolved over time to have a wide variety of functions Need to understand protein structure and ultimately function Genome: entire DNA/RNA complement of an organism Constant, static same whether it is within liver or lung cells What is different is the proteomes The entire protein complement Think of it as within an organism or within individual cells. We can frequently deduce the function of a protein by its amino acid sequence Part of the ability to predict the function of a protein by its amino acid sequence comes from its molecular structure. Molecular structure has a hierarchy. Primary Structure: sequence of amino acids Secondary structures: the way the primary structure folds due to the sequence of amino acids. Tertiary structure: the highest order structure and of proteins, is folded from secondary structure even further. Tertiary structures can then bond together to form quaternary structures. Proteins with multiple subunits or multimeric proteins Can do a wide variety of functions This protein structures can be involved in the regulation of the activity of proteins Some of these proteins are structural components of cells or in the movement of cells, organelles in the cell, communications. 1 Primary Structure: sequence of amino acids Secondary Structure: once these amino acids are transcribed and pasted together, the string or portions of the string of amino acids will fold into alpha helices, beta sheets, dictated by linear arrangement of amino acids. Tertiary Structure: Once the secondary structures have been completed, the protein further folds and arranges itself into this. Involves the arrangement of secondary structure into regions of domains. If the proteins are multimers, these proteins interact and form quaternary structures. Primary Structure: ● The sequence of amino acids and the polypeptide ● Amino acids have a carbonyl group, central carbon, amino group, a hydrogen and a unique R group ● Amino acids come together by generation of polypeptide bond that forms between the carbonyl group of one amino acid and the amino group of the other. ● When a variety of amino acids are put together, you get a polypeptide. ○ always a free carboxyl at Cterminus and a free amino group at the Nterminus ● One property that gives the protein its unique function are the R groups of the amino acids within the chain. Peptide: 2030 AA Polypeptide: up 4000 Protein: complex of polypeptides or a polypeptide Terms used indiscriminately Secondary Structure: ● Results of folding of the localized parts of the polypeptide chain or protein into various spacial arrangement 2 ● A single polypeptide chain can exhibit multiple types of secondary structure or multiple numbers of a given secondary structures ● Predominant secondary structures are: alpha helix, beta sheets, and beta turns ● Spontaneously form in defined regions of the polypeptide chain by the formation of hydrogen bonds between certain residues ○ allows the backbone to form the defined structures ● Alpha Helix ○ carbonyl oxygen atom is hydrogen bonded to aminehydrogen atom towards the Cterminus. ○ this bonding between the carbonyl oxygen and the amine hydrogen at these defined spatial arrangement that gives the helical structure. ○ Structure has directionality ○ All R groups face out of the helix. ○ R groups determine if the chain is hydrophilic or hydrophobic. ● Beta Sheet ○ consist of laterally stacked Beta strands of AA ○ can be AA where the orientation is parallel or antiparallel ○ usually made up of 58 AA residues ○ directionality 3 ● Beta Turn ○ usually composed of 34 AA residues ○ generally located at the surface of a protein ○ redirect the polypeptide backbone into the interior ○ stabilized by hydrogen bonding ○ certain AA found in turns than in sheets and helices ■ glycine ■ proline bulky Rgroup that generally induces a bend ○ Loops are larger than beta turns. Tertiary Structure ● overall conformation of the polypeptide, the 3D structure ● stabilized by the hydrophobic interactions between nonpolar side chains and and hydrogen bonding between polar R groups and peptide bonds. ○ keep the secondary structures within a confined tertiary structure ● bonds are a bit weaker than those in secondary structure. ● Within the definition of tertiary structures there are defined regions called motifs ○ combination of secondary structures that contribute to tertiary structures. ○ Variety of signature motifs, those that have particular functions ○ EF Hand: Helixloophelix motif ■ binds to calcium ○ Zincfinger: helix and two sheets ■ binds to zinc in a binding pocket ■ two histadines and cystines that are spaced in such a way that when the protein is folded, it creates the pocket for zinc binding ■ binds to DNA and RNA ○ Coiledcoil motif ■ hydrophobic R groups are found in regular, repeated heptet sequences in alpha helices ■ fibrous proteins and involved in self adhesion 4 ■ amphipathic: two different sides, one hydrophobic, one hydrophilic ■ allows two or three alpha helices to wind around each other ● Another level of structure is the domain ○ larger order structures ○ Hemagglutinin protein ■ center is globular domain composed of alpha helices and beta sheets ■ other portion is elongated and looks fibrous ■ structural feature that can be acidic or proline rich, and can do a variety of things ■ and different domains have different functions within a protein ■ within domains can be a variety of motifs Quaternary Structure ● only found where there is more than one polypeptide in the mature protein molecule ○ hemagglutinin: trimeric protein ● interaction and arrangement of the individual subunits Domains can be modularly put together depending on what the mature function of the protein will be. Not part of hierarchy structure but the most advanced structure are the macromolecular machines. these are made up of a number of proteins (3040 or more) that come together and interact and perform some sort of function. Proteins that serve similar functions are evolutionarily conserved. Globin family: hemoglobin (a tetramer), myoglobin, and leghemoglobin all three of these carry oxygen, despite the differences in their tertiary structures. Part II: Function of a protein is dictated by its structure. ● critical to a protein’s function is that it properly folds. ● cell has mechanisms to promote the folding of proteins; to ensure proper folding and if it misfolds, to dispose of those proteins. ● Improperly folded proteins do not have appropriate biological activity ○ can function as a null protein ○ can initiate diseases ○ whole group of diseases characterized as improper folded protein diseases. ○ disease is initiated by the accumulation of improperly folded proteins. ● Most proteins are able to, under different circumstances, find several different conformations. ○ within a cell, a protein will have one conformation. ○ this is the native state and is the most stable conformation (usually). ○ has to do with energenics. ● Under certain conditions, such as changes in pH, temperature, etc, proteins can lose their native state and therefore, their biological activity. (Denaturation) ○ disrupts the noncovalent interactions that hold the protein structure together 5 ○ Denatured proteins under certain conditions can return to their native state (renaturation). ○ This indicates that in general, the amino acid sequence of a protein is most of the time sufficient to allow the protein to spontaneously fold into its native state. ● The cell has a variety of mechanisms to assist in the proper folding. ● THe folding of proteins is facilitated by molecules called chaperones. ○ Two Families of Chaperones: ■ Molecular Chaperones: molecules that bind to and stabilizes unfolded and partially folded proteins. ● allows prevention of of degradation of protein before it is properly folded. ● facilitates the proper folding of proteins. ● Hsp70; binds to a newly synthesized polypeptides in an ATP dependent manner ○ ATP is required!! ● folds the protein and the folding requires the energy from ATP hydrolysis (ATP to ADP) to assist in the folding. ■ Chaperonins: ● Molecular Chaperones ○ Hsp70 ○ As the proteins come off of the ribosome (as they are translated) or are released in the cytoplasm after translation, they can bind to Hsp70 ○ only if Hsp70 is bound to ATP, which changes Hsp70’s conformation. ○ The binding of the unfolded protein is ATP dependent, ○ ATP binds to one region of Hsp70, and the unfolded protein binds to the substrate binding domain. ○ ATP is hydrolysed to ADP, and an inorganic is released. ■ this promotes the folding of the protein by changing the conformation of Hsp70 that binds to the protein. ● The hydrolysis of ATP to ADP facilitates the folding of a protein and the release of an inorganic phosphate. ○ ADP is then exchanged for ATP ■ ADP is released from Hsp70 and is replaced by ATP ■ This exchange of ADP to ATP facilitates the release of the folded protein from Hsp70 6 ● The chaperonins are molecular machines. ○ GroEL (bacterial) ● Either as it’s being released from the ribosome or is a partially folded/misfolded protein in the cytosol, it enters the large macromolecular machine chamber. ○ composed of a variety of molecules ○ ATP is hydrolysed and the cap will be placed on. ○ ATP is added and join the chaperonin ○ With a second hydrolysis of ATP, another cap is added and the proper folding occurs, which is released with the hydrolysis of ATP and ADP and the cap is removed. ○ Protein is released. ● The principles between the chaperones and chaperonins are the same, the difference is: ○ Chaperones are individual molecules 7 ○ Chaperonins are molecular machines (made up of a variety of molecules and subunits and are rather large. ○ What is required of both is energy is required to properly fold the protein molecules. ● In addition to proper folding, virtually all proteins at one point or another are chemically modified after synthesis. ○ these modifications can alter their activity, lifespan, and their localization in cells. ○ can occur at the free carboxy or amino termination or on a variety of side chains. ● A common modification is acetylation. ○ occurs at the free amino terminus ○ About 80% of proteins are acetylated during their lifetime. ○ influences the stability of proteins. ● There are a variety of chemical modifications. ○ Phosphorylation: the primary amino acids that can be phosphorylated are serine, tyrosine, histidine, and threonine ■ signal transduction pathways: signals are transduced through cells by the phosphorylation and dephosphorylation of serine, threonine, and tyrosine ○ Hydroxo group addition to amino acid proline: found primarily in collagen (connective tissue molecules). ○ Methylation: can include histidine, lysine, or arginine ○ Gamma carboxyglutamate: carboxylation of amino acids, primarily glutamate ○ Proteins that will be secreted or attached to the outside of a cell can undergo glycosylation. ■ the addition of linear or branched carbohydrates to cells. Protein Degradtion As proteins become damaged, they can fail to function properly. Protein lifespan varies from a few minutes to the life of the cell. There are several pathways for the degradation of proteins. ● Lysosomal: membrane bound organelle 8 ○ interior is highly acidic and contains hydrolytic enzymes ○ primarily degrade extracellular proteins taken up by the cell. ○ Endocytosis: the uptake of relatively small molecules, some soluble or membrane associated by vesicles. ■ an invagination of the plasma membrane. ■ Mature into early endosomes, which mature into late endosomes, and ultimately fuse with the lysosomes of the cell to form secondary lysosomes. ● where the molecules taken up are broken down into building blocks, which can be recycled. ○ Phagocytosis: development of membrane bound organelle but the uptake is larger, such as bacteria. ■ development of membrane bound organelle. ■ invagination of membrane and then the pinching off of the membrane, and a phagosome is formed. ● a single lipid bilayer ■ merges with primary lysosomes to form the secondary lysosome for degradation. ○ Autophagy: the degradation of cellular components, ■ a mechanism in which cells can recycle materials already present in the cell. ■ self eating ■ part of the ER membrane will encircle an organelle to be degraded to form a double lipid bilayer called an autophagosome and that merges with the primary lysosomes into the secondary lysosome for degradation. ● Proteasomedirected degradation: macromolecular machine that degrades proteins that have been polyubiquitinated ○ Ubiquitin76 amino acid polypeptide that is covalently attached to lysine residues. ○ Polyubiquitin chain is recognized by proteasome ○ degrades protein in an ATPdependent manner into short (78 amino acid) peptides. ○ Macromolecular machine ○ Ubiquitination and ProteasomeMediated Protein Degradation ○ The process of ubiquitination involves at least three steps that involve three different enzymes ■ E1, E2, E3. ■ E1: ubiquitin activating enzyme ■ E2: ubiquitin conjugating enzyme ■ E3: ubiquitin ligase. ■ E1, using ATP, allows for the addition using the hydrolysis of ATP for the addition of ubiquitin to the E1 enzyme itself, activating the process. ■ Ubiquitin is transferred from E1 to E2, covalently attached. ■ E3 then ligates the ubiquitin and conjugates it to a lysine on the target protein. ■ Process repeats multiple times and the protein is then recognized by the proteasome and as part of the hydrolysis of ATP, the protein is broken down into peptides and the ubiquitin is released to be recycled and reused. Lecture on Parts I and II Protein domains ● strings of amino acids and some tertiary structure interactions create structures called domains 9 ● Domains often carry out specific functions ● Protein families shared evolutionarily conserved domains, and thus, common functions ● Protein domains are structural units of proteins that often perform different functions. Proline breaks alpha helixes Part III: Enzymes and Chemical Work of Cells Any proteins are able to do their function because they interact with a number of different cellular molecules. ● can be proteins, lipids, nucleic acids, etc. ● Molecules that they interact with are referred to as ligands. ● When talking about proteins, specifically enzymes, or any kind of interactions between proteins and another molecule, a term that is important is specificity ○ the ability of a protein to bind to one molecule in preference to another. ○ in order to get defined work done in a cell, whether it’s transport or something else, there has to be some sort of specificity in the interactions. ● Affinity: the strength of binding of a particular binding of a molecule to a particular protein. ○ dissociation constant: Kd, the measure of affinity of a protein ligand constant. The stronger the interaction, the lower the Kd. ○ All interactions is an equilibrium process. The skew depends on the affinity between the molecules in question. ○ The higher the affinity, the stronger the binding and the lower the dissociation constant. AntibodyAntigen Interactions are Highly Specific ● When your body sees a bacteria, B cells secrete antibodies that are specific for certain components on them. ● At the region where light and heavy chains interact, there is a recognition site for that antigen. ● The antigen recognition site is based on the ability of the molecule that is recognized to be able to fit into that pocket. ● The better it does so, the higher the affinity. Enzymes ● Catalyzed chemical alteration of their ligands ○ ligand = substrate ● Most chemical reactions in the cell are catalyzed by enzymes ● enzymes do not alter the reaction, do not change the products. ● Enzymes increase the reaction rate by lowering the activation energy ○ accelerate the formation of products from reactants without altering the value of delta G. ● Enzymes act on a substrate and change its composition. 10 In the reaction in which there is no catalyst, the activation energy of the transition state is large. All the enzyme does, is lowers it. Enzyme Active Site Binds the Substrate. ● must have a binding site to bind to the substrate. ● Protein Kinase A (PKA). ○ a protein with a large and small domain with a binding pocket ○ in the binding pocket, it interacts with the target peptide ○ a kinase ■ a kinase is a protein that phosphorylates other proteins ■ transfers a phosphate to another protein ■ must bind to ATP to do this. ■ must have in its binding site, a region to bind ATP and a region to bind the substrate that will be phosphorylated. Conformational Change Caused by Substrate Binding Within the small domain, is the glycine lip. In PKA there is the small domain with the glycine lip and the large domain. When the substrate binds, and it brings ATP together with the peptide, resulting in a conformation change and bringing the glycine lip closer and sort of closes of the active site. Enzyme Kinetics Conversion of one substrate into a product using two different enzyme concentrations and increasing the substrate concentrations. Kinetics, the rate of reactions, and how they occur for enzymes and membrane transport proteins. Two Important parameters 1. Vmax: that max velocity of enzyme reaction at saturating substrate concentration. 11 a. if you have a given concentration of enzyme, as you increase the concentration of substrate, the rate of formation of product for a given enzyme concentration, you reach a maximum velocity at which the enzyme cannot work any faster. 2. Km: Michaelis constant. The measure of affinity of an enzyme for its substrate. a. determined as the substrate concentration that yields half maximal reaction volume. b. Half the Vmax c. The lower the Km, the higher the affinity. d. As you increase the substrate concentration, you reach the V max for that concentration. e. The Km, or affinity, is half of the V max. f. If you have one fourth the concentration, the Km is still half the V max, which is lowered. g. The V max is dependent upon the enzyme concentration, however, the Km is independent of the enzyme concentration. h. Increasing the enzyme concentration, does not increase the Km. It is a measure of affinity. i. V max is a measure of the maximum velocity of at which product can be created and that does change with enzyme concentration. ● Certain enzymes can work on more than one substrate. ○ There will be differences. ● Conversion of one substrate into a product using constant enzyme concentration and two substrates. ● Enzyme has two different affinities for the two substrates. ● If you have a substrate with a high affinity, it is going to reach at the same enzyme concentration a higher Vmax, and a lower Km. ● If it is a low affinity substrate, you will eventually reach the same V max, but the concentration that it takes to get to half V max will be greater. Thus, Km will be higher. ● The Vmax is the same for either substrate. The Km will differ because it is the measure of affinity. ○ The substrate with the greater affinity will have the lower Km. 12 Enzymes in Common Pathways Usually located in close proximity to each other. Diffusion of product to next enzyme in the pathway. A scaffold keeps enzymes close to each other Multifunctional Enzyme enzyme encoded by a single gene that has more than one catalytic activity. Pyruvate Dehydrogenase Complex Pyruvate is acted upon and acetyl CoA is created. carries on three sequential enzymatic reactions. Part IV: Mechanisms for Regulating Protein Function ● There are a variety of mechanism for regulating protein functions. ● An important aspect of protein function is allostery. ○ a change in a protein’s tertiary structure induced by the addition of a ligand ○ ligand can be an inhibitor, activator, substrate, etc. ○ when the ligand binds, the protein undergoes a conformational change, and therefore the protein’s function changes. ● A common protein that undergoes allosteric change is hemoglobin. ○ carries oxygen in RBC ○ cooperative bonding of oxygen ■ a type allostery ○ allows multisubunit proteins to respond more efficiently to small changes in ligand concentration ○ can be positive or negative ○ positive occurs in hemoglobin, the initial binding of oxygen molecule enhances or changes the conformation of hemoglobin so that the binding of subsequent oxygen molecules occurs more rapidly or with greater affinity ○ Negative affinity would be the binding of the first molecule would inhibit the binding of other like molecules. ○ We can see it is positive with hemoglobin with the binding the initial oxygen molecule, you get a very rapid increase in the percent saturation. Ligand Binding Activates Protein Kinase A Catalytic Subunit. 13 ● An allosteric example ● PKA within cells is normally inactive and a tetramer, composed of two catalytic subunits and two regulatory subunits ○ The regulatory subunit has a domain in it called a pseudosubstrate domain which binds in the substrate binding site of the catalytic domain and it inhibits the activity of the kinase by preventing the binding of the substrate. ○ PKA is activated by a small second messenger called cyclic AMP (cAMP). ○ cAMP binds to the regulatory subunits of PKA and induces a conformational change. ○ This conformational change releases the catalytic subunits and the catalytic subunits can now bind to the substrate and to ATP and go about phosphorylating their substrates. ○ Whatever the signal is that activate PKA stops, the level of cAMP goes down and is released from the regulatory subunits, and will go back to an inactive conformation of PKA ○ cAMP is a molecule that induces allosteric change by inducing a change in the conformation of PKA. A positive cooperativity. ■ small changes in cAMP induces large changes in PKA. Calmodulin Functions as Ca2+ Binding Switch Protein The binding of divalent Ca ions can induce allosteric changes. GTPBinding Proteins Function as Molecular Switches ● Gproteins ● Function as molecular switches ● Bind to GTP or GDP and depending on which of these are bound to, changes whether the protein is on or off. ● Guanine nucleotide binding proteins, GTPases ● Large number of these. ● Function in a wide variety of cellular processes. ● In general, if the protein is bound to GTP, it is in an on conformation and if it is bound to GDP, it is in the off conformation. ● The difference in the single phosphate, from tri to diphosphate, changes the conformation and changes its activity. 14 ● In a cell where there is no signals coming in, the GTPases are in the off position. ● A signal comes in and activates a regulatory protein for the GTPases called the guanine exchange factor or GEF and it will exchange the GDP for GTP. ○ The GTPase will now be bound to GTP and changed to conformation into the on conformation. ○ GAPs are proteins that catalyze the hydrolysis of GTP to GDP so that the GTPase undergoes an allosteric change to the off conformation. Regulation of Protein Function by Phosphorylation/Dephosphorylation Kinases use ATP and they transfer a phosphate to the protein. (Bind to the R group) The phosphorylation mostly activates a protein. A signal comes into the cells, activating the protein kinase and the protein is phosphorylated. Phosphatase removes the phosphate from a protein, and inactivates it. Chapter Ten: Biomembranes and Cell Architecture Lipid Composition and Structural Organization ● What differs between prokaryotic and eukaryotic cells is that eukaryotic cells have membrane bound organelles, are generally larger than prokaryotes organelles are generally surrounded by one or more lipid bilayer ● Each organelle is unique in their protein composition, the lipids in their membranes, and each carry out specific and unique functions. ● They are able to carry out much more complex work because they are able to sequester different pathways and different types of work in different organelles. ● Having these membranes allows cells to keep what’s supposed to be inside the organelles inside the organelles and what is outside of the cell outside of the cell. ● By the incorporation of proteins of the membranes allows the cell to determine what can enter and leave the cell. ● Cytoskeleton gives the cell its shape and positions the organelles in the cell. Bilayer Structure of Biomembranes 15 ● Exterior of cell: ectoplasmic side ● Interior of cell: cytosolic side. ● Lipid bilayer is composed of two phospholipid layers. ○ polar hydrophilic heads ○ nonpolar hydrophobic, fatty acyl chains. ■ sequestered between the layers of polar heads. ○ Contributes to the behavior of the membrane ○ Hydrophobic tails create a barrier against hydrophilic molecules. ● Provides a level of stability ○ spontaneously form in a watery environment by hydrophobic and van de Waal interactions ● Lipid membranes are dynamic structures ○ can change shape ○ seen in endo and exocytosis. ○ and the fusion of these with the target membrane. ● Integral membrane proteins: are across the entire membrane. ● Lipid anchor proteins: associate with lipids, have a lipid moiety resemble fatty acyl chains. Do not go through the whole membrane. ● Peripheral membrane proteins: associate with other proteins or hydrophilic heads, can be found on either side. Faces of Cellular Membranes ● faces are a nomenclature that says what the sides of the membrane faces. ● Plasma membrane ● Ectoplasmic face: outside contents, within the organelles ● Cytosolic face: cellular side. ● This assigning of faces allows us to keep straight the orientation of the membranes and its contents. ● The inside of a vacuole is referred to as the ectoplasmic face because when it merges with the plasma membrane, the inside is what faces the outside of the cell. Classes of Lipids found in Membranes Three major classes of lipids found in biomembranes 1. Phosphoglycerides a. the most abundant b. also referred to as phospholipids. c. amphipathic d. phosphatidylcholine i. most common e. phosphatidylethanolamine f. phosphatidylinositol i. Found only on the outer leaflet of plasma membranes ii. Glycosylation 16 g. phosphatidylserine i. mechanisms of cell death ii. found on the inner leaflet of plasma membrane h. Tail groups: fatty acyl tails, associated with each with hydrophobic interactions and van der Waal interactions i. Derived through glycerol3phosphate j. 1618 carbons long k. Carbon tails can have 03 double bonds l. named for the head group. 2. Sphingolipids a. composed of sphingosine i. amino alcohol b. most common is sphingomyelin 3. Steroles a. Cholesterol b. ring structure c. amphipathic 17 Lipids and some proteins are mobile in membranes ● are able to move laterally in membranes. ● Discovered by fluorescence recovery after photobleaching Lipid Diffusion Indicates Membranes are Fluidlike ● fluidity is determined by temperature ○ at lower temperature, membranes are more gel like ○ at higher temperatures, these interactions are looser. Factors Affecting Bilayer Fluidity 1. Lipid Composition a. Which lipids are present 2. Structure of Hydrophobic Tails a. long, saturated tails, (no double bonds) tend to have a gel like composition b. Short unsaturated tails tend to have a more fluid like composition c. Or if there are double bonds, those double bonds introduce a kink that allows the interactions to not be so rigid, allowing for fluidity. 3. Temperature 4. Cholesterol Content a. tends to get into the lipid bilayer and at normal concentration, tend to inhibit the random movement of head groups and decreases the fluidity b. at lower concentrations, creating a more fluid movement. c. can also affect the width of the lipid bilayer. Lipid Composition of Different Cellular Membranes ● Different membranes have different percentages of the different lipids. ● As a result, they all behave differently 18 Factors Affecting Lipid Composition ● Site of synthesis of various lipids ○ phospholipids (PL) Endoplasmic reticulum ○ Sphingolipids golgi apparatus ● Cell type/Function ○ Intestinal epithelial cells ■ basolateral face contacts other epithelial cells and other underlying structures ■ apical face faces the lumen of the gut ● higher concentration of sphingolipids than the basolateral face ● thought to increase the stability of the membrane through hydrogen bonding. ● involved in the absorption of nutrients ■ have very different percentages of lipids in the various sides Bilayer Distribution of Membrane Lipids ● Different phospholipids are usually more abundant in one one leaflet of the membrane than the other. ● sphingomyelin and phosphatidylcholine are more abundant on the outer leaflet ● Phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are more abundant in the inner leaflet. ● Cholesterol is usually evenly distributed between the two leaflets. ● Can do a variety of things such as curvature of the membrane. How Does Asymmetry of Lipid Distribution Arise? ● Flipflopping of lipids is energetically unfavorable ○ hydrophilic, polar head would need to pass through the hydrophobic interior of the membrane ○ Extremely difficult ○ Flippases enzyme that can move certain phospholipids from one leaflet to the other in an ATP dependent manner. ● May result in part from the site of lipid synthesis ○ Sphingomyelin synthesized on luminal (exoplasmic) face of Golgi ■ becomes part of exoplasmic face of the cell membrane ○ Phosphoglycerides synthesized on the cytosolic face of the ER ■ this face is identical in composition to the cytosolic face of the cell membrane. Certain lipids can transfer by budding off the ER membrane into droplets. single layer of membrane Cholesterol and trigylcerides Lecture on Parts III and IV, Beginning of Chapter Ten Enzymes lower the transition state Vmax is dependent on the enzyme concentration 19 The affinity of the substrate for the enzyme is not dependent upon the concentration on the enzyme but the conformation of the substrate and enzyme and how closely they fit molecularly What sort of reactions they have, how strongly they bond in the site. A cell has 4 copies of enzyme Y. Each molecule Y. Can convert substrate S to product P at a rate of 10 molecules/minute. At saturating substrate concentration, what is the Vmax? 40 molecules/minute What is the Km of the reaction? 20 molecules/minute In response to need, the cell has made more molecules of enzyme Y (7) what is the Vmax? 70 molecules/minute What is the Km? 35 molecules/minute Enzyme Y can work on two different substrates, S and S’. It will convert them o P and P’ respectively Would the Vmax differ for the two substrates? Why? No, because the enzyme can only work so fast Would the Km be different? Yes, there would be different affinities between the two substrates and the enzyme because one will fit better in the substrate binding domain. Km is the substrate concentration it takes to get to half the Vmax. Dependent on substrate affinity, not concentration Multifunctional enzymes are frequently in the same pathway. Amphipathic: will have a polar and Nonpolar region Know the types and their precursor (the molecules they’re made out of). All phosphoglycerides have fatty acyl chains and a polar head. These heads vary between the types. The difference between saturated and unsaturated is saturated makes the layer more rigid. The double bond(s) in the unsaturated chain creates a more fluid layer. Ectoplasmic side: faces outside the cell Cytosolic side: faces the cytosol of the cell. Glycosylation only occurs on the exoplasmic side of the cell, the outside of the cell. Chapter Ten: Biomembranes and Cell Architecture Lipid Composition and Structural Organization Protein Components of Membranes and their Function 20 Membrane proteins are classified based on membraneprotein interactions 1. Integral Membrane Proteins: a. transmembrane proteins b. Span the entire phospholipid bilayer c. Three segments: i. cytosolic domain ii. exoplasmic domain iii. membranespanning domain d. most are glycosylated on the exoplasmic face. e. cytosolic and exoplasmic domains are generally hydrophilic f. membrane spanning domains are composed of hydrophobic amino acids with the side chains pointed out. i. They interact with hydrocarbons of the phospholipid bilayer ii. Membrane spanning domains are usually alpha helices, but can be composed of multiple beta sheets. g. Some transmembrane proteins can move laterally in the membrane, others cannot due to their connection to the cytoskeleton. 2. LipidAnchored Membrane Proteins a. Have hydrophobic hydrocarbon chains attached to the protein covalently bound b. Bound covalently to lipid molecules in the membrane c. The bond to hydrocarbon chains is reversible i. can come on and off. 3. Peripheral Membrane Proteins a. bound to the membrane indirectly, usually through interactions with integral membrane proteins b. can interact with polar head groups 4. Glycosylation is only found on the exoplasmic face of the cell. Structure of a Transmembrane Protein Glycophorin A ● found on RBC ● 2 subunits, but each has 1 membrane spanning domain (alpha helix, 2025 hydrophobic Amino acids) ○ extracellular side with glycosylations ○ cytosolic domain ○ 2 units associate together to form a coilcoiled motif which creates a diamer ■ homodimer (total homo) 21 Integral Membrane Proteins with Seven Membrane Spanning Domains Includes: Gprotein coupled receptors Members of this class have the same structures: 7 alpha helices that cross the membrane with regions on either side. IonChannels Multipass Transmembrane Proteins protein complexes ion channels are generally tetrameric each individual subunit has 2 membrane spanning alpha helices that ultimately associate to form a channel the amino acids facing the exterior of these fatty acyl chains are hydrophobic while the inside is hydrophilic Most transmembrane proteins’ membrane spanning domains are alpha helices. Porins: unusual transmembrane proteins found in bacteria and the outer membranes of chloroplasts and mitochondria provides channels for small, watersoluble molecules such as sugars or water itself. composed of tetramers of identical subunits membranespanning domains are composed of beta sheets. 22 inside is hydrophilic and outside is hydrophobic LipidAnchored Membrane Proteins Can be on the exoplasmic face or the cytosolic face. GPI Anchor found on the exoplasmic side proteins are covalently attached to phosphatidylinositol GPI: glycosylphosphatidylinositol Acylation found on cytosolic side myristate or palmitate attached to glycine Prenylation found on cytosolic side farnesyl or geranylgeranyl attached to cysteine 23 Glycosylated Proteins Glycosylation: addition of carbohydrates (sugar chains) to a protein most transmembrane groups have carbohydrate chains covalently attached to them Carbohydrate chains are always attached to the exoplasmic domain of the protein Lipids can also be glycosylated Glycolipids are also only found on the exterior leaflet of the membrane Carbohydrate groups interact with extracellular matrix, growth factors, antibodies, etc. Carbohydrates are added to proteins and that affects their properties (the protein’s) glycosylation is important in blood typing The A, B, O, and AB groups are different carbohydrates attached to RBCs depending on the composition of the Carbohydrates determines the recognition as different antigens There are specific motifs that allow proteins to interact at the membrane lipid binding motifs Pleckstrin Homology (PH) Domain most common associates with phosphatidylinositol Common Features of Cell Membranes Allowing water to pass through the membrane to maintain equilibrium is not efficient 1. Osmosis a. the direction of flow depends on the solution surrounding the medium b. Isotonic solution: concentration of solutes is the same outside the cell as it is inside. There is no net movement of water c. Hypotonic solution: concentration of solutes is greater inside the cell than outside, causing water to move in and causes the cell to swell. d. Hypertonic Solution: concentration of solutes is greater outside the cell than inside, causing water within the cell to move out and shrivelling it up. 24 2. Junctions between cells: a. adherence b. celltocell communication 3. Cell surface receptor a. bind signalling molecules Organelles of the Eukaryotic Cell Each compartment has specialized functions and specialized features some have single lipid bilayers, others have double. Single Membrane Endosomes function to take up soluble macromolecules from the cell exterior Clathrincoated pits: invaginates and pinches from the membrane to form membranebound vesicle Vesicle is delivered to early endosome the processing of contents begins Endosome delivers membranes and contents to lysosomes for degradation Lysosomes acidic organelles containing hydrolytic enzymes (acid hydrolysases) found only in animal cells, degradation Autophagy: degradation of old organelles and other cell components by the lysosome Endocytosis: uptake of small proteins and soluble macromolecules by the cell Phagocytosis: uptake of large, insoluble particles by the cell, e.g. bacteria Hydrolytic enzymes are important in degradation Peroxisomes degrade fatty acids and other toxic compounds Oxidases: enzymes that use molecular oxygen to oxidize substances results in the formation of H2O2 (toxic) Catalase: degrades H2O2 into H2O and O2 Endoplasmic Reticulum important in the synthesis of lipids, membrane proteins, and secreted proteins Smooth ER: site of synthesis of fatty acids and phospholipids most cells have very little smooth ER Abundant in hepatocytes (liver cells) specialized enzymes modify (detoxify) harmful chemicals (pesticides, carcinogens) Rough ER: site of synthesis of membrane, organelles, and secreted proteins ribosomes attached to cytosolic surface of the ER membrane secreted proteins enter the lumen of the ER Golgi Complex: membrane bound and secreted proteins synthesized in the ER vesicles containing these proteins bud from ribosome free regions of the ER and fuse to the Golgi complex Three defined regions: Cis Medial Trans Vesicles fuse with the cis region and progress to the medial region and then to the trans specialized enzymes in each region modify the proteins depending on the structure of the protein and its final location whether its membrane or secreted 25 Vesicles from the ER fuse to the cis Golgi, proteins move from cis to medial, proteins move from medial to trans, vesicles bud from trans Golgi and direct proteins to the membrane, cell surface , or lysosomes Double Membrane Nucleus: outer lipid. bilayer is continuous with the ER. There is a space between the inner and outer layers along the outer bilayer there are a series of pores that material can enter or leave the nucleus through nuclear pores within the nucleus is our DNA in the form of chromosomes Different regions of the nucleus serve slightly different functions despite the lack of further physical division Mitochondria outer membrane, inner membrane and between them is the intermembrane space. continuous with the inner membrane are invaginations that pass into the center into the mitochondrial matrix these are cristae Chloroplasts outer and inner membrane structures thylakoids site of photosynthesis and where ATP is generated Mitochondria and chloroplasts contain their own DNA which is circular Chapter Eleven: Membrane Transport membrane transport are the specific mechanisms cells have to move materials in and out. most of the membrane transport involves membrane transport proteins that are transmembrane or integral membrane proteins very few things can cross the membrane by diffusion Passive Diffusion an Ineffective Means of Transport Across the Membrane This is due to the large hydrophobic region of the membrane Gases are permeable Small uncharged polar molecules like ethanol are permeable Small uncharged polar molecules like urea and water and slightly permeable Large, uncharged polar molecules, ions, and charged polar molecules are all impermeable 26 Classes of Membrane Transport Proteins 3 Classes: 1. ATPPowered Pumps: 10^0 to 10^3 ions/s 2. Ion Channels: 10^710^8 ions/s 3. Transporters: 10^210^4 molecules/s a. Uniporters b. Symporter (cotransporter) c. Antiporter (cotransporter) What’s common to all is during the transport, there is a conformational change that regulates the movement of materials back and forth. Can facilitate movement of molecules at rates much higher than that of diffusion. 27 ATPPowered Pumps move cargo against the concentration gradient (low to high) requires ATP to do so hydrolysis Ion Channels move cargo with the concentration gradient water, ions, small molecules high concentration to low concentration facilitated diffusion: does not require an input of energy can be gated or ungated ungated are open all of the time or most of the time gated open and close when acted upon by certain stimuli Transporters Three types: uniporter, symporter, antiporter Uniporter: single molecule down its concentration gradient (high to low) Symporter: molecules end up on the same side of the lipid membrane cotransporter: transports more than one molecule/ion. Uses the movement of the energetically favorable movement of one ion/molecule to power the movement of the other. Antiporters: cotransporter molecules move in opposite direction Uniporters transport small, hydrophilic molecules 28 Glucose transported by GLUT1 receptor in RBC Transport is characterized by high rate of transport compared to diffusion Transport not affected by degree of hydrophobicity Protein complexes facilitate transport diffusion occurs over the entire surface of the membrane limited number of transporters at high concentration gradients, transport occurs at maximum rate (velocity V max) resembles enzyme kinetics Uniports are specific for a single molecule or closely related molecules transporter has a measurable affinity Km concentration of substrate that results in transport at half the maximal rate Unidirectional Transport of Glucose by GLUT1 Transporter Glucose binds to the receptor, it changes the conformation. The glucose is released and the release changes the conformation again, this time back to the original outward facing position. as it is being transported, glucose is being modified, changing the receptor’s affinity for it and preventing it from going back over. ATPPowered Pumps 29 transmembrane proteins have at least one ATPbinding site of cytosolic face of the pump Couples hydrolysis of ATP to transport molecules uses the energy to move the molecules against the concentration gradient. Function to maintain proper ionic conditions within the cytoplasm or within the organelles. generate and maintain concentrations across the membrane Four classes of ATPpowered pumps PClass Pump Plasma membrane of plants, fungi, bacteria (H+ pump) Plasma membrane of higher eukaryotes (Na+/K+ pump) Apical plasma membrane of mammalian stomach (H+/K+ pump) Plasma membrane of all eukaryotic cells (Ca2+ pump) Sarcoplasmic Reticulum membrane in muscle cells (Ca2+ pump) Composed of two alpha subunits and two beta subunits during transport, one of the alpha subunits becomes phosphorylated. VClass Pumps 30 FClass Pump ABC Superfamily 31 Flippase important in drug resistance ABCB1 (MDR1) is a protein that is expressed in adrenal, kidney, and brain tissue that exports lipophilic drugs. Lecture January 28 Passive Transport: does not use membrane transporter, some nonpolar Facilitated Transport: transports things across the membrane, do not need ATP Active Transport: the use of ATP Ca2+ Transport into the SR of Muscle Cells Pclass pump SR is similar to ER stores calcium under certain signals, calcium is pumped into the lumen and that initiates muscle contraction In the resting state, the transporter has two binding sites for calcium and the alpha subunit is unphosphorylated and has an ATPbinding site. 1. Calcium binds to the transporter and ATP binds to the alpha subunit. (from cytosol). 2. Phosphorylation of an aspartate on the alpha subunit due to phosphate transfer from the ATP 3. ADP is released and that changes the conformation of the receptor 4. This opens the face into the lumen and calcium can move into the receptor 5. The alpha subunit is phosphorylated 6. Conformation then changes back to the original resting state conformation. this goes against the concentration gradient. Na+/K+ Transport pclass pump phosphorylation of the alpha receptor Transports 2 molecules/ions in opposite directions. 32 1. Resting state with binding of Na+ and ATP 2. Phosphorylation of the aspartate residue 3. The phosphorylation induces a conformational change 4. The conformational change alters the opening which releases the Na+ and takes on K+ binding. 5. Dephosphorylation occurs, which induces another conformational change, allowing for the release of K+ into the cytosol. E1 conformation: opening faces the cytosol E2 conformation: opening faces the lumen. VClass H+ ATPases Found in vacuoles, lysosomes, and endosomes Maintain acidic pH within organelles Contain a hydrophobic domain and a hydrophilic domain hydrophilic domain is on the cytosolic side 1. ATP binds to the domain on the cytosolic side 2. Hydrolysis provides the energy for the transport of H+ across the membrane a. there is no phosphorylation/dephosphorylation b. moving against the concentration gradient Protons alone do not create an acidic environment Positive charges on the inside of the membrane will attract negative charges on the outside of the membrane and create an electric potential. Will also need a channel that will move negative ions inside the organelle. this movement of negative ions into the organelle alongside the movement of H+ prevents the buildup of electrical potential and gets the pH to be acidic. 33 ABC Transporter E. col i Lipid Flippase 2 large molecules with ATPbinding domains on the cytosolic side homologous to MDR1 or multidrug resistant protein 1 involved in chemotherapeutic drug resistance in cancer ATP is hydrolyzed and material is moved from the inside of the cell to the outside of the cell. 34 Cotransport by Symporters and Antiporters mechanism for transporting molecules against their concentration gradients. Couples energy produced from transport of a molecule down its concentration gradient (energetically favorable) to transport another molecule against its concentration gradient (energetically unfavorable). Examples: Electrochemical gradient of Na+ is used to transport glucose or amino acids into cells Na is the cotransported molecule Glucose or amino acids are transported against high concentration gradients Cotransport: transport is coupled neither molecule can be transported without the other. Na+/Glucose Cotransport symporter: both molecules are being transported in the same direction 1. outward facing conformation has opening to exterior 2. binding of ion changes the conformation 3. ions are deposited and the conformation reverts back to the outward facing opening. 35 Cardiac Muscle Cells: Na+/Ca2+ Antiporter cation antiporter increase in cytosolic Ca2+ induces contraction lowering cytosolic Ca2+ reduces strength of the contraction Cytosolic Ca2+ pumped out against a high concentration gradient 3 Na+ pumped into the cytosol and 1 Ca2+ is pumped out. Nongated Ion Channels Allow ions to move through the membrane down their concentration gradient. generates electrical potential across the membrane. important in transducing a variety of signals. If a membrane is freely permeable: total concentration of + and When equal, there is no net flow and no electrical potential If you have a membrane permeable to say sodium, sodium moves across and that creates a charge separation. Sets up a membrane electric potential The same would happen if it were permeable to K+ or on the opposite side. 36
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