Chapter 3

1 Chapter 3 Protein Structure and FunctionBroad functional classesSo Proteins have structure and Fine!-Why do we care to know more????Understanding functional architechture gives usPOWERto: Diagnose and find reasons for diseases Create modifying drugs Engineer our own designer-proteinsDNA(mRNA)Translation:Tr anslation into 3D structure:3D structure determines function:Modifications:Chemical modification of aminoacidsInteraction with other moleculesProteolytic cleavage(Location)New 3D structureNew functionaProteins are single, unbranched chains of amino acid monomersaThere are 20 different amino acidsaThe amino acid sidechains in a peptide can become modified, extending the functional repetoire of aminoacids to more than hundred different amino protein s amino acid sequence determines its three-dimensional structure (conformation) aIn turn, a protein s structure determines the function of that proteinaConformation (=function)

2 Is dynamically regulated in several different waysProtein structure determines functionAll amino acids have the same general structure but the side chain (R group) of each is different C R:Hydrophilic:BasicAcidicNon-chargedHydr ophobic Special Hydrophilic amino acidsHydrophobic and special amino acidsBackboneSide-chainsPeptide bonds connect amino acids into linear chainsSide chain modifications change the chemical (functional) properties of proteinsGlycosylationUbiquitylation=> Expanding the repetoire of existing amino acid side-chains to > 100 variations!AcetylationPhosphorylationHyd roxylationMethylationCarboxylationFour levels of structure determine the shape of proteinsaPrimary:the linear sequence of amino acids peptide bondsaSecondary:the localized organization of parts of a polypeptide chain ( , the helix or sheet)backbone hydrogen bondsaTertiary:the overall, three-dimensional arrangement of the polypeptide chainhydrophobic interactions, hydrogen bonds (non-covalent bonds in general) and sulfur-bridgesaQuaternary:the association of two or more polypeptides into a multi-subunit complexPrimary and secondary structure (example.)

3 Hemagglutinin) -strand -helixSecondary structure Helix Sheet (U)-turnMotifs are regular combinations of secondary structures. Motifs form domains!Three examples of Motifs from different types of DNA-binding proteinsTertiary structureStructural, functional or topological domains are modules of secondary and tertiary structureGlobular domainTertiary structureEach of these proteins contain the EGF globular But each of these proteins have a different functionTertiary structureDifferent graphical representations of the same protein(tertiary structure)Quaternary structureMultiprotein complexes: molecular machinesSequence homology suggests functional and evolutionary relationships between proteinsWhen the stucture of a newly discovered protein is known, comparison to other proteins across species can help predict functionFolding, modification, and degradation of proteinsThe life of a protein can briefly be described as.

4 Synthesis, folding, modification, function, newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final proteinaAll molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the moleculeaMost proteins have a limited lifespan before they are degraded (turn-over time)Aberrantly folded proteins are implicated in slowly developing diseasesAn amyloid plaque in Alzheimer s disease is a tangle of protein filamentsThe information for protein folding is encoded in the sequenceFolding of proteinsin vivo is promoted by chaperonesLarge proteins with a lot of secondary structure may require assisted folding to avoid aggregation of unfolded protein- Molecular chaperones and chaperonins prevent aggregation of unfolded proteinFolding of proteinsin vivo is promoted by chaperonesLarge proteins with a lot of secondary structure may require assisted folding to avoid aggregation of unfolded protein- Chaperones and chaperonins prevent aggregation of unfolded proteinFunctional

5 Design of proteinsaProtein function often involves conformational changesaProteins are designed to bind a range of molecules (ligands)`Binding is characterized by two properties: affinity and specificityaAntibodies and enzymes exhibit precise ligand/substrate-binding specificityBut can have variable affinitiesaEnzymes are highly efficient and specific catalysts`An enzyme s active site binds substrates(ligands) and carries out catalysisAntibody/antigen interaction: an example for ligand-binding with high affinity and specificityEnzymes have high substrate affinity sites and catalytic sitesKinetics of an enzymatic reaction are described by Vmaxand KmKinetics of an enzymatic reaction are described by Vmaxand KmEnzymes in one pathway can be physically associatedMechanisms that regulate protein activityaAltering protein synthesis rate and proteasomal degradationaAllosteric transitions`Release of catalytic subunits, active inactive states, cooperative binding of ligandsaChemical modification: `Phosphorylation, acetylation etc.

6 Dephosphorylation, deacetylation activationaCompartmentalizationProtein degradation via the ubiquitin-mediated pathwayCells contain several other pathways for protein degradation in addition to this pathwayATPA llosteric transitions: Cooperative binding of ligandsSigmoidal curve indicates cooperative binding (of ligands, substrates, ca ions) in contrast to standard Michaelis-Menten KineticsConformational changes induced by Ca2+binding to calmodulinCooperativebinding of calcium: binding of one calcium enhances the affinity for the next calciumWhen 4 calcium are bound a major allostericconformational changeoccursCalmodulin is a switch proteinbecause this effect in turn regulates other proteins bound by the compact calmodulinAnother class of switch proteins: GTPasesChemical modificationExample: Phosphorylation dephosphorylationProteolytic cleavage of proinsulin to produce active insulinCompartmentalizationExample.

7 Membrane proteinsaEach cell membrane has a set of specificmembrane proteins that allows themembrane to carry out its activitiesaMembrane proteins are either integralor peripheralaIntegral transmembrane proteins containone or more transmembrane helicesaPeripheral proteins are associated withmembranes through interactions withintegral proteinsSchematic of membrane proteins in a lipid bilayerMechanisms that regulate protein activityaAltering protein synthesis rate and proteasomal degradationaAllosteric transitions`Release of catalytic subunits, active inactive states, cooperative binding of ligandsaChemical modification: `Phosphorylation, acetylation etc. dephosphorylation, deacetylation activationaCompartmentalizationExample containing all levels of regulatin of protein activityGFP-tagged GLUT4 Now that you KNOW the basic principles of protein structure and function you can UNDERSTAND:Protein and ProteomeAnalytical techniquesPurifying, detecting, and characterizing proteinsaA protein must be purified to determine its structure and mechanism of actionaDetecting known proteins can be usefull for diagnostic purposesaMolecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties (size, mass, density, polarity, )`Elementary toolbox includes.

8 Centrifugation, electrophoresis, liquid chromatography (LC), spectrometry, ionization/radiation. -applied in various advanced forms and separate molecules that differ in mass or densityElectrophoresisseparates molecules according to their charge:mass ratioSDS-polyacrylamidegel electrophoresisEven coating of proteins allows even charge distribution -> larger mass = higher total chargeTwo-dimensional electrophoresis separates molecules according to their charge and their massHighly specific enzymes and antibody assays can detect individual proteinsImmunoblot (= Western Blot) based on affinityLiquid chromotography (LC):Separation of proteins by size: gel filtration chromatographyAdd mobile phase: bufferStationary phase:Separation of proteins by charge: ion exchange chromatographyAlso.

9 Reversed-phase LC: separation by hydrophobicityStationary phase: non-polar, Mobile phase: moderately polarSeparation of proteins by specific binding to another molecule: affinitychromatographyProteomics, the analysis of complex protein mixturesaGenome databases allow prediction of genes -> protein primary structureaEach protein can be fragmented into peptides which are composed of aa aa has a unique mass to charge ratio at a given pHaEach protein therefore has a unique peptide-fingerprintaTechnique: proteins->peptides->mass/charge ratio measurement -> compare against whole proteome (genome based) database -> identify proteinsTime-of-flight mass spectrometry measures the mass of proteins and peptidesMatrix-Assisted-Laser-Desorption /Ionization Time-of-flight mass spectrometry (MALDI-TOF MS)MS spectrumExample of a proteome analysis workflowCell/tissue of interestIsolate organelles (fractionation)Confirm organelle-specific proteinsSubfractionate, detect peptides, identify corresponding proteinsX-ray crystallography is used to determine protein structureOther techniques such as cryoelectron microscopy and NMR spectroscopy may be used to solve the structures of certain types of proteins