Nonenzymatic Protein Function & Protein Analysis PDF

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Chapter Summary: pg. 105

Ch. 3 – Nonenzymatic Protein Function & Protein Analysis 3.1 Cellular Functions 

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Typical functions within the cell: o Supporting cellular shape & organization o Acting as enzymes Structural & motor proteins are found: o In abundance within individual cells o In ECM

1. Structural Proteins o Cytoskeleton: 3D web or scaffolding system for the cell  Comprised of proteins that are anchored to the CM by embedded protein complexes o Intracellular + Extracellular support for the tissues of the body  Extracellular matrices composed of proteins  Tendons, ligaments, cartilage, and basement membranes are all proteinaceous o Structural proteins generally have highly repetitive secondary structure & a supersecondary structure (motif)  Super-secondary structure: a repetitive organization of secondary structural elements together  Sometimes referred to as a motif  This regularity gives many structural proteins a fibrous nature o Primary structural proteins: a) Collagen  Has a characteristic trihelical fiber o 3 left-handed helices woven together to form a secondary right-handed helix  Makes up most of the ECM of connective tissue  Found throughout the body  Important in providing strength & flexibility  Importance of structure of collagen is highlighted in the disorder osteogenesis imperfecta (brittle bone disease) o Collagen = mjr component of bone  Forms a unique & specific secondary helical structure based on the abundance of the aa glycine  Replacement of glycine with other aa’s can cause improper folding of the collagen protein & cell death, leading to bond fragility b) Elastin  Important component of ECM of connective tissue  Primary role: to stretch & then recoil like a spring o Restores the original shape of the tissue c) Keratins  Intermediate filament proteins found in epithelial cells  Functions: o Contribute to mechanical integrity of the cell o Regulatory proteins o Primary protein that makes up hair & nails d) Actin  Makes up microfilaments & the thin filaments in myofibrils  Most abundant protein in euk cells  Have polarity  have a positive side & a negative side o Allows motor proteins to travel unidirectionally along an actin filament e) Tubulin  Makes up microtubules o MTs are important for providing: i. Structure

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ii. Chromosome separation in mitosis & meiosis iii. Intracellular transport with kinesin & dynein Has polarity o Negative end of MT is usually located adjacent to the nucleus o Positive end of MT usually in periphery of a cell

2. Motor Proteins o Some structural proteins also have motor functions in the presence of motor proteins  Motile cilia & flagella of bacteria & sperm  Contraction of the sarcomere in muscle o Display enzymatic activity  Act as ATPases that power the conformational change necessary for motor function o Responsible for muscle contraction & cellular movement o Have transient interactions with either actin or MTs o Include: a) Myosin  Primary motor protein that interacts with actin (thick filament in a myofibril)  Can be involved in cellular transport  Each myosin subunit has a single head & neck o Movement at the neck is responsible for the power stroke of sarcomere contraction b) Kinesins  Associated with microtubules  2 heads – at least 1 remains attached to tubulin at all times  Play key roles in: o Aligning chromosomes during metaphase o Depolymerizing MTs during anaphase of mitosis  Important for vesical transport in the cell o Bring vesicles toward the positive end of the MT (opposite polarity of dyneins)

Stepwise Activity of Kinesins Kinesins move along MTs in a stepping motion such that 1 or both heads remain attached at all times.

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c) Dyneins  Associated with microtubules  2 heads – at least 1 remains attached to tubulin at all times  Involved in the sliding movement of cilia & flagella  Important for vesical transport in the cell o Bring vesicles toward the negative end of the MT (opposite polarity of kinesins) Classic example of motor proteins’ polarities in NEURONS  Kinesins bring vesicles of NT to the positive end of the axonal MTs (toward the synaptic terminal)  Dyneins bring vesicles of waste or recycled NT back toward the negative end of the MT (toward the soma) through retrograde transport

3. Binding Proteins o Proteins primarily exert enzymatic or structural functions within the cell

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BUT they also can have stabilizing functions in individual cells & the body  These proteins transport or sequester molecules by binding to them Include:  Hemoglobin  Calcium-binding proteins  DNA-binding proteins (often txn factors)  Others Each has an affinity curve for its molecule of interest (e.g., oxyhemoglobin dissociation curve)  Curve differs depending on the goal of the binding protein  Sequestration of a molecule: the binding protein usually has high affinity for its target across a large range of concentrations so it can keep it bound at nearly 100%  Transport protein: must be able to bind or unbind its target to maintain steady-state concentrations; likely to have varying affinity depending on the env’tal conditions 

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4. Cell Adhesion Molecules (CAMs) o Proteins found on the surface of most cells o Aid in binding the cell to the ECM or other cells o # of diff types – all are integral membrane proteins o Classified into 3 major families: a) Cadherins  A group of glycoproteins that mediate calcium-depending cell adhesion  Often hold similar cell types together (e.g., epithelial cells)  Diff cells usually have type-specific cadherins o Epithelial cells use E-cadherin o Nerve cells use N-cadherin b) Integrins  A group of proteins that all have 2 membrane-spanning chains ( a & b) Many medications o Chains v important in binding to & communicating with the ECM target selectins &  Play v important role in cellular signaling integrins.  Can greatly impact cellular function by promoting cell division, apoptosis, or Research has shown other processes that ability of cancer cells to  Examples of integrin functions: metastasize (break o Integrin a IIbb3 allows platelets to stick to fibrinogen (clotting factor) away from a tumor  Causes activation of platelets to stabilize the clot & invade other o WBC migration distant tissues) is o Stabilization of epithelium on its basement membrane associated with o Other processes unique expression c) Selectins patterns of CAMs. By targeting these  Unique – they bind to carbohydrate molecules that project from other cell CAMs, metastasis surfaces may be avoided.  These bonds are the weakest formed by the CAMs To stop the clotting  Expressed on WBCs & endothelial cells that line blood vessels process during  Play an important role in host defense (like integrins) o Including inflammation & WBC migration

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5. Immunoglobulins o Immune system = v complex & made up of many diff&types of cells & proteins WBC Migration Using Selectins Integrins  These cells & proteins have a common purpose: to rid the body of foreign invaders o Antibodies (Immunoglobulins, Ig)  Most prominent type of protein found in immune system  Proteins produced by B-cells  Function to neutralize targets in the body (i.e., toxins & bacteria) & then recruit other cells to help eliminate the threat o Structure:  Y-shaped proteins  Made up of 2 identical heavy chains & 2 identical light chains  The heavy & light chains are held together by: a) Disulfide linkages b) Noncovalent interactions  Each Ab has an antigen-binding region at the tips of the “Y”  Within this region, there are specific polypeptide sequences that will bind 1 (& only 1) specific antigenic sequence  Constant region = the remaining part of the Ab molecule  Involved in recruitment & binding of other cells of the immune system (i.e., macrophages) o When Abs bind to their targets (antigens), they can cause 1 of 3 outcomes: a) Neutralizing the antigen, making the pathogen or toxin unable to exert its effect on the body b) Opsonization: marking the pathogen for destruction by other WBCs immediately c) Agglutinating (clumping together) the antigen & Ab into large insoluble protein complexes that can be phagocytized & digested by macrophages

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3.2 Biosignaling  

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Biosignaling: a process in which cells receive & act on signals Proteins participate in biosignaling in different capacities, incl. acting as: 1. Extracellular ligands 2. Transporters for facilitated diffusion 3. Receptor proteins 4. Second messengers The proteins involved can have functions in either: 1. Substrate binding 2. Enzymatic activity

1. Ion Channels o Proteins that create specific pathways for charged molecules o Classified into 3 main groups that have different mechanisms of opening, but all permit facilitated diffusion of charged particles  Facilitated diffusion: a type of passive transport; the diffusion of molecules down a concentration gradient through a pore in the membrane created by this transmembrane protein (ion channels)  Used for molecules that are impermeable to the membrane (large, polar, or charged)  Allows integral membrane proteins to serve as channels for these substrates to avoid the hydrophobic FA tails of the phospholipid bilayer o 3 main groups: a) Ungated Channels  No gates  unregulated  All cells possess potassium channels o Net efflux of potassium ions through these channels unless potassium is at eqbm b) Voltage-Gated Channels

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Gate is regulated by the membrane potential change near the channel Many excitable cells (i.e., neurons) possess voltage-gated sodium channels o Closed under resting conditions o Membrane depolarization causes a protein conformation change that allows them to quickly open & then quickly close as the voltage increases  Voltage-gated non-specific sodium-potassium channels are found in cells of the SA node of the heart o Serve as pacemaker current  As the voltage drops, these channels open to bring the cell back to threshold & fire another AP c) Ligand-Gated Channels  The binding of a specific substance or ligand to the channel causes it to open or close o NTs act at ligand-gated channels at the postsynaptic membrane  Inhibitory NT g-aminobutyric acid (GABA) binds to a chloride channel & opens it  Activity at the neuromuscular junction & most chemical synapses relies on ligand-gated ion channels  NS esp. makes use of this type of gating Km & vmax parameters that apply to Es are also applicable to transporters such as ion channels in membranes  The kinetics of transport can be derived from the Michaelis-Menten & LineweaverBurk eqns  Km = the [solute] at which the transporter is functioning at ½ its maximum activity  

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2. Enzyme-Linked Receptors o Membrane Rs may also display catalytic activity in response to ligand binding o Have 3 primary protein domains: a) Membrane-spanning domain – anchors the receptor in the CM b) Ligand-binding domain – stimulated by the appropriate ligand & induces a conformational change that activates the catalytic domain c) Catalytic domain – activated by induced conformational change (^)  Often results in initiation of a second messenger cascade o Classes: a) Receptor tyrosine kinases (RTK) (classic example)  Composed of a monomer that dimerizes upon ligand binding o Dimer = active form that phosphorylates additional cellular enzymes, including the receptor itself ( autophosphorylation) b) Serine/threonine-specific protein kinases c) Receptor tyrosine phosphatases 3. G Protein-Coupled Receptors (GPCRs) o A large family of integral membrane proteins involved in signal transduction o Characterized by their 7 membrane-spanning a-helices o Rs differ in specificity of the ligand-binding area found on the extracellular surface of the cell o Heterotrimeric G proteins: utilized by the GPCRs in order to transmit signals to an effector in the cell  Named for their intracellular link to guanine nts (GDP & GTP)  Binding of a ligand increases the affinity of the receptor for the G protein  Binding of the G protein represents a switch to the active state & affects the intracellular signaling pathway  Several diff G proteins that can result in either stimulation or inhibition of the signaling pathway

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3 main types of G proteins : a) Gs – stimulates adenylate cyclase (increases levels of cAMP in the cell) b) Gi – inhibits adenylate cyclase (decreases levels of cAMP in the cell) c) Gq – activates phospholipase C o Cleaves a phospholipid from the membrane to form PIP2 o PIP2 then cleaved into DAG & IP3 o IP3 can open calcium channels in the ER, increasing calcium levels in the cell

Trimeric G Protein Cycle (Gs or Gi)

3 subunits comprise the G protein: a) a subunit b) b subunit c) g subunit  Trimeric G Protein Cycle:  Inactive form: binds GDP & is in complex with b & g subunits 1. When ligand binds to the GPCR, R becomes activated and (in turn) engages the corresponding G protein 2. Once GDP is replaced with GTP, the a subunit is able to dissociate from the b & g subunits o Activated a subunit alters the activity of adenylate cyclase  If the a subunit is as  enzyme is activated  If the a subunit is ai  enzyme is inhibited 3. GTP on the activated a subunit is dephosphorylated to GDP 4. The a subunit will rebind to the b & g subunits, rendering the G protein inactive 3.3 Protein Isolation 

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In order to better understand a specific protein, it’s important to be able to isolate the protein for study Great amt of variation in the physical & chemical properties of proteins o These differences can be exploited in order to purify the protein of interest Proteins & other biomolecules are isolated from body tissues or cell cultures by cell lysis & homogenization (crushing, grinding, or blending the tissue of interest into an evenly mixed solution o Centrifugation can then isolate proteins from much smaller molecules before other isolation techniques must be employed Most common isolation techniques:

1. Electrophoresis  1 of most important analytical techniques in molecular biology  Works by subjecting compounds to an electric field, which moves them according to their net charge & size  Negatively charged compounds migrate toward the positively charged anode  Positively charged compounds migrate toward the negatively charged cathode  Migration velocity (v): the velocity of this migration o Directly proportional to the electric field strength (E) & to the net charge on the molecule (z) o Inversely proportional to a frictional coefficient (f)  Depends on the mass & shape of the migrating molecules

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Polyacrylamide gel: the standard medium for protein electrophoresis  Slightly porous matrix mixture – solidifies at room temperature  Proteins travel through this matrix in relation to their size & charge  Gel acts like a sieve, allowing smaller particles to pass through easily while retaining large particles o A molecule will move faster through the medium if it’s: a) Small b) Highly charged c) Placed in a large electric field o Molecules will migrate slower (or not at all) when they’re: a) Bigger & more convoluted b) Electrically neutral c) Placed in a small electric field  Size of a standard polyacrylamide gel allows multiple samples to run simultaneously

Electrophoresis uses an electrolytic cell (DG > 0, Ecell < 0). Anions always move toward the anode & cations always move

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As an electrolytic (nonspontaneous) cell, electrophoresis moves charged particles toward their respective oppositely charged electrodes; the larger the particle, the more slowly it migrates.

Types: a) Native PAGE o Polyacrylamide gel electrophoresis (PAGE): a method for analyzing proteins in their native states o Limited by the varying mass-to-charge & mass-to-size ratios of cellular proteins bc multiple different proteins may experience the same level of migration o Functional native protein can be recovered from the gel after electrophoresis, but only if the gel hasn’t been stained  Most stains denature proteins o Most useful to compare the molecular size or the charge of proteins known to be similar in size from other analytic methods like SDS-PAGE or size-exclusion chromatography b) SDS-PAGE o Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

 SDS: a detergent that disrupts all noncovalent interactions Separates proteins on the basis of relative molecular mass alone Starts with premise of PAGE but adds SDS  SDS binds to proteins & creates large chains with net negative charges  Neutralizing the protein’s original charge & denaturing the protein o As the proteins move through the gel, the only variables affecting their velocity are E (electric field strength) & f (frictional coefficient; depends on mass) o After separation, the gel can be stained so the protein bands can be visualized & the results recorded c) Isoelectric Focusing o Proteins separated on the basis of their isoelectric point (pI)  pI = the pH at which the protein or aa is electrically neutral, with an equal # of positive & negative charges  Zwitterion: electrically neutral form for individual aa’s o Amino group is protonated o Carboxyl group is deprotonated o Any side chain is electrically neutral  For polypeptides, the pI is primarily determined by the relative #s of acidic & basic aa’s o Exploits the acidic & basic properties of aa’s by separating on the basis of isoelectric point (pI)  A protein stops moving when pH = pI o Procedure: i. Mixture of proteins is placed in a gel with a pH gradient  Acidic gel at the positive anode  Basic gel at the negative cathode  Neutral in the middle ii. Electric field is generated across the gel  Positively charged proteins begin migrating toward the cathode  Negatively charged proteins begin migrating toward the anode iii. As the protein reaches the portion of gel where the pH is equal to the protein’s pI, the protein takes on a neutral charge & will stop moving o Example scenario: i. Start with protein with pI of 9  When protein is in an env’t with pH of 9, it will carry no net charge ii. If place protein onto gel at pH of 7, there’ll be more protons around the protein iii. Protons will attach to the available basic sites on the protein, creating a net positive charge on the molecule iv. Net positive charge will cause protein to be attracted to the negatively charged cathode (located on basic side of the gradient) v. As protein moves closer to the cathode, the pH of the gel slowly increases vi. Eventually, as protein nears pH of 9, the protons creating the positive charge will dissociate  The protein will become neutral again o Quick way to remember the pH of each end of the gel:  Associate acids with protons, which carry a positive charge  positively charged anode o o

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Associate bases with the negatively charged hydroxide ion  negatively charged cathode

2. Chromatography  Uses physical & chemical properties to separate & ID compounds from a complex mixture  Refers to a variety of techniques that require the homogenized protein mixture to be fractionated through a porous matrix  Isolated proteins are immediately available for ID & quantification  In all forms, the concept is identical: the more similar the compound is to its surroundings (by polarity, charge, and so on), the more it will stick to & move slowly through its surroundings  Preferred over electrophoresis when large amts of protein are being separated  Procedure: a) Place the sample onto a solid medium (stationary phase or absorbent) b) Run the mobile phase through the stationary phase o Will allow the sample to run through the stationary phase ( elute) c) Depending on the relative affinity of the sample for the stationary & mobile phases, different substances will migrate through at different speeds o Components that have a high affinity for the mobile phase will migrate much ...