Membrane Protein Function BCH210H F24 Lecture Notes PDF

MEMBRANE PROTEIN FUNCTION STRUCTURAL CHANGES HELP WITH FUNCTION… Sian Patterson, Ph.D. Associate Professor, Teaching Stream  Learning Objectives By the end of this lecture, students should be able to: Explain how the structure of a membrane protein is...

MEMBRANE PROTEIN FUNCTION STRUCTURAL CHANGES HELP WITH FUNCTION… Sian Patterson, Ph.D. Associate Professor, Teaching Stream  Learning Objectives By the end of this lecture, students should be able to: Explain how the structure of a membrane protein is crucial for its ability to move ions, molecules or transmit signals across the membrane. Describe the essential features of the main types of signalling pathways. Identify the role lipids and enzymes play in transmitting signals across the membrane and throughout the cell. Distinguish between passive and primary vs. secondary active transport. 2  membrane protein 5HT2A Serotonin Receptormolecules has different interactal Drug/hormone ayer attached it droxyl- edge binding to - cholesterol my ⑧ R (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate is What happens after binding? ~ onintera 3  SIGNALING Cellular Signalling can bind outside All physiological processes involve biochemical interactions and reactions, allowing a cell to carry out its function and adapt. conformatioa The binding of signaling molecules (ions, hormones, sugars) to their receptors initiate processes such as metabolic pathways and gene expression. Proteins are essential for carrying out this response but must be regulated. Copyright © 2014 by Nelson Education Ltd. 4  Signal Transduction transmission 10 message Signal transduction cascades have many components in common: protein Binding specifically to a signalling molecule in response to a physiological stimulus. Reception of the message by the receptor, 20 messengers usually an integral membrane protein. (cAMP , (a2 +) Relay of the primary message to the cell interior by the generation of an intracellular secondary messenger. Amplification and transduction of the signal. Response then termination of the signal cascade. 5  G-Protein-Coupled Receptors (GPCRs) structure GPCRs contain 7 TM segments. common among many protein types They are part of super-family of membrane proteins. in GPT Conformational changes release the G proteins. bound Copyright © 2014 by Nelson Education Ltd. State Can bind many different ligands: Natural : serotonin, epinephrine, prostaglandins, dopamine, psilocin/psilocybin Kdvalus different Synthetic : morphine, histamine, LSD Cligands& receptors < kind of lik km) *** Binding is key for GPCR specificity. *** 6 ↓ kd = Strong binding  Characterizing Binding Interactions Non-covalent interactions (ionic bonds, hydrogen Top view bonds, van der Waals interactions) between the amino acid side chains and molecule’s functional transmembrane ligand a groups influences the binding affinity. Binding affinities can be used to characterize and compare the non-covalent interactions between two biomolecules (proteins, ligands, cofactors, substrates, drugs, etc.). a a side chain Kd values are dissociation constants, a lower Kd kon equals stronger binding. A + B ⇄ AB on certain amount bound koff Remember, binding is saturable based on stoichiometry and reversible for non-covalent Kd = [A][B] = koff = Ka-1 interactions. [AB] kon 7 don't need equations  🔗 The b2-Adrenergic Receptor Brian Kobilka and co-workers set out to solve the structures of the b2-adrenergic receptor in the inactive and active states ligand epinephrine : β2-Adrenergic Receptor Required 20 years of protein 206 The N engineering and hard work to TTM “see” the answer Brian Kobilka and co-workers solved the structure of the receptor in the inactive and active states. 179 Ligand binding induces small changes in TM5 on the extracellular side. huge A 14 Å movement in TM6 transmits the signal inside.↑ 10-10 m releasea protein Major conformational changes in TM6 promote the release of Gα-GTP, which activates adenylyl cyclase to produce the secondary messenger cAMP. cAMP can activate other enzymes, including Protein Kinase A (PKA), a transferase that can phosphorylate and activate/inactivate other enzymes. Kobilka, B. (2012) Nobel Lecture “The Structural FIGURE 5. Basis A comparison of the carazolol-bound, of G Protein Coupled Receptor inactive-state structure of t Signaling”. 8 (gray) and the active-state structure of the β2AR (green) from the β2AR-Gs co  epinephrine signals for fatbreakdown What's the most effective way to turn off epinephrine signalling? A) cAMP breakdown > - AMP B) Epinephrine unbinding most effective (start of pathway) C) GTP hydrolysis > - GDP D) Protein Kinase A inactivation > - PKA A , C , D also must happen  protein (GPCR) Ras Proteins type of G Members of the superfamily of small GTPases that bind and hydrolyze GTP. > - GDP Activated in numerous signalling pathways that initiate cell proliferation and is is apoptosis. not if entrolled signalling there A conformational change can be seen in the switch I and switch II motifs upon phosphate release (GTP → GDP). Defects in GTP hydrolysis can lead to uncontrolled signalling and cancer. 10 involved in cancer signalling COURSES Signalling and Human Health nature.com/articles/d41586-018-05267-x Defects at any point along the pathway can lead to disease. DiM PKA-P Post-translational modifications and conformational changes play a key role in these pathways. Mutations in the receptors or effector proteins can prevent ligand-receptor interactions or the protein-protein interactions needed for activation/inactivation. Knowing the structure of (membrane) proteins is important to understand their function. Drugs can also be designed to bind and inhibit or stimulate the proteins involved in signalling to modulate the cellular response. 11  insulin Two other important types of signalling as Enzyme-linked Receptors: Usually contain a single phosphory transmembrane segment that may be homodimers or dimerize enzyme portion upon ligand binding. eg. insulin auto- tyr-kinases (PTMs) Activation leads to auto-phosphorylation or phosphorylation by tyrosine kinases. Examples: insulin, epidermal growth factor (EGF), Jak/STAT. stuk in membrane breakes Phospholipid mediated Signalling: Phospholipases hydrolyze phospholipids to produce other 2nd messengers like messenger 2n diacylglycerol (DAG) or IP3 leading to the release of calcium from the ER. Examples: Eicosanoid and AKT signalling. 12  Hormone vs. Hormone – who will win?!? Insulin and epinephrine are competing hormones. I have competing roles (slovde vsbreakdowna Phosphorylation of the Insulin Receptor Substrate (IRS)-1 and activation of the pathway also leads to phosphorylation of the β-adrenergic receptor by Protein Kinase B (PKB). many downstream This post-translational modification effects regulate leads to internalization and what happen degradation, terminating GPCR signalling. can no longer intiate fat break dow 13 insulin is stronger hormone  TRANSPORT Membrane Transport Small, uncharged or lipophilic molecules may cross by passive diffusion (slow & concentration dependent). Transport is essential for life: Nutrients in - garbage out. Inorganic ions in and out. Cofactors https://makeagif.com/i/GUOwjZ Integral membrane proteins are important for transport via: Facilitated diffusion. Active transport with/without ATP. 14 Sort the following molecules based on their permeability across the membrane from most to least permeable: zwitzerton (+ 1-) Alanine 4. / Protons + H. 3 more with X membrane protei Oxygen 1.: Water 2 can cross Leaquaporin/. uncharged ; has dipole 15  Facilitated Diffusion is Saturable Facilitated transport is dependent on the presence Saturable of binding sites on saturable Rate of transport (v) membrane proteins. The rate of transport (v) is saturable at high substrate Concentration dependent concentration (ie. all binding sites are occupied). A hyperbolic curve is similar to what is seen for simple Copyright © 2014 by Nelson Education Ltd. catalytic enzymes. 16  Channel Proteins Membrane transporters that facilitate diffusion are also known as (ion) channel proteins. The structure of the membrane protein is key for its function. variety of structure must have many TMs , Important features of ion channels: pos neg size Selectivity (K+ vs. Cl- or K+ vs. Na+). charg Rapid conductance of ions (108 / sec). Can be gated (open/closed) due to stimuli. Copyright © 2014 by Nelson Education Ltd. 17  closed Potassium Ion Channel Dr. Roderick Essential for many cellular processes: MacKinnon Regulation of cell volume Secretion of hormones Electrical impulse formation (esp. neurons) site Each subunit contributes a selectivity filter of 5 amino acids (TVGYG) that contribute to K+ binding. flinding ↑ it Filter 4 backbone carbonyls and the Thr side-chain hydroxyl bind the K+ ions. (their oxygen) oxygen Changing the sequence alters the selectivity for other cations. 18  Gating the Potassium Channel In response to specific stimuli (voltage gating/intracellular pH change), helix bending at a conserved Gly residue occurs in the regulatory domain. Gly99 acts as a molecular hinge to open/closed (gate) the channel. helices can bend + k TMs w Copyright © 2014 by Nelson Education Ltd. many 19  Active Transport Active transport is the movement of molecules against their concentration gradient. In primary active transport, the breakdown of ATP, light energy, or the passing of electrons generates energy for transport. Secondary transporters use the gradient of one molecule to power the formation of another (eg. Na+-glucose transporters). diff direction - same direction 20 Conformational Change in a Flippase MsbA – a bacterial lipid transporter  Key Messages Conformational change is key for transport and signalling but is also essential for the regulation of activity of membrane proteins and downstream signalling enzymes. Non-steroidal receptors are integral membrane proteins with a variety of structures (single TM vs. multi-pass), resulting in the creation of secondary messengers for protein activation/inactivation in the cell. Transport can be general or specific based on the structure of the membrane protein and is either concentration dependent or based on the availability of ATP or a co-transporter. 22