Chapter 3 Proteins: Chapter 3 Proteins:

Shape, Structure, and Shape, Structure, and FunctionFunction

Proteins Execute Cell Proteins Execute Cell FunctionsFunctions

► EnzymesEnzymes► Channels and pumpsChannels and pumps► Signal MoleculesSignal Molecules► MessengersMessengers► Molecular MachinesMolecular Machines► Structural Support Structural Support ► Cell RecognitionCell Recognition

Protein Shape and StructureProtein Shape and Structure

Peptide Bond Links Amino Acids into Polypeptide Chain

Protein Shape and Structure Protein Shape and Structure ► Evolution fine-tuned structure and Evolution fine-tuned structure and

chemistrychemistry► Shape dictated by amino acid sequenceShape dictated by amino acid sequence

polypeptide backbonepolypeptide backbone

side chainsside chains

Protein Shape and StructureProtein Shape and Structure

Sequence Determines Structure

Protein Shape and StructureProtein Shape and Structure

Weak Noncovalent Bonds/Interactions important to the folding of polypeptide chain

Protein Shape and StructureProtein Shape and Structure

► Fold into Conformation of Lowest Fold into Conformation of Lowest EnergyEnergy

► Common Folding PatternsCommon Folding Patterns

alpha helixalpha helix

Beta SheetBeta Sheet

Coiled CoilsCoiled Coils

Protein Shape and StructureProtein Shape and Structure

Levels of organization protein structureLevels of organization protein structure► primary= aa seqeunceprimary= aa seqeunce► secondary= stretches of alpha helix, beta sheetssecondary= stretches of alpha helix, beta sheets► tertiary=3d organizationtertiary=3d organization► quartenary=complete structure of protein w/ > 1 quartenary=complete structure of protein w/ > 1

poly-peptide chainpoly-peptide chain

Protein Shape and StructureProtein Shape and Structure► Protein Domain= Fundamental Unit of OrganizationProtein Domain= Fundamental Unit of Organization

independently folding unitindependently folding unit

40-350 aa modular unit; combine to form larger 40-350 aa modular unit; combine to form larger proteinsproteins

different domains have different functionsdifferent domains have different functions► Fold= central core of domain; comprised of beta sheets and Fold= central core of domain; comprised of beta sheets and

alpha helices in various combinations; limited numberalpha helices in various combinations; limited number

Short signature sequences identify homologous protein domains

Protein Shape and StructureProtein Shape and Structure

Percentage of total genes in respective genomes containing one or more copies of a particular protein domain

Domain shuffling during the course of evolution

Protein Shape and StructureProtein Shape and StructureProtein ModuleProtein Module► Smaller than an average domain, generally 40-200 Smaller than an average domain, generally 40-200

aaaa► Particular versatile structuresParticular versatile structures► Easily integrated into other proteins; form parts of Easily integrated into other proteins; form parts of

many different proteinsmany different proteins

Protein Shape and StructureProtein Shape and Structure

Protein Families EvolvedProtein Families Evolved► similar 3d structuresimilar 3d structure► portions or aa sequence conservedportions or aa sequence conserved► non-conserved portions impart new functionalitynon-conserved portions impart new functionality

serine proteasesserine proteases

homeodomain proteinshomeodomain proteins

kinaseskinases

immunoglobulinsimmunoglobulins

Protein Shape and StructureProtein Shape and Structure

► Sequence Homology SearchesSequence Homology Searches► Amino Acids Sequence Threading Amino Acids Sequence Threading ►Modules form parts of many different Modules form parts of many different

proteinsproteins

Protein Shape and StructureProtein Shape and Structure

Protein Shape and StructureProtein Shape and Structure

Larger proteins can assemble from identical monomeric subunits

Protein Shape and StructureProtein Shape and Structure

► Larger proteins often contain more than one Larger proteins often contain more than one polypeptidepolypeptide

► Proteins can serve as subunits for assembly of large Proteins can serve as subunits for assembly of large structuresstructures

► Self AssemblySelf Assembly

Protein FunctionProtein Function► Function of protein dictated by physical interactions w/ other Function of protein dictated by physical interactions w/ other

moleculesmoleculesspecificity and ligand affinity governed by multiple weak specificity and ligand affinity governed by multiple weak noncovalent bonds noncovalent bonds

active/binding site often cavity on protein surface formed by neighboring active/binding site often cavity on protein surface formed by neighboring aa aa or aa that may belong to different portions of polypeptideor aa that may belong to different portions of polypeptide

Protein FunctionProtein Function

Conformation determines chemistryConformation determines chemistry► Regions adjacent to active or ligand binding site may restrict water to Regions adjacent to active or ligand binding site may restrict water to

increase ligand bindingincrease ligand binding► Clustering of polar or chged residues can alter chemical reactivityClustering of polar or chged residues can alter chemical reactivity► Type and orientation of exposed aa side chains govern chemical reactivityType and orientation of exposed aa side chains govern chemical reactivity

Protein FunctionProtein Function

““Evolutionary tracing” to determine sites critical to protein Evolutionary tracing” to determine sites critical to protein functionfunction

► 3d structure of protein family members are similar even when aa 3d structure of protein family members are similar even when aa homology falls to 25%homology falls to 25%

► Map unchg aa or nearly unchg from all known family members onto Map unchg aa or nearly unchg from all known family members onto 3d structure of one family member3d structure of one family member

► Most invariant positions often on surface and represent ligand Most invariant positions often on surface and represent ligand binding sitebinding site

Protein FunctionProtein Function

Proteins Bind to other Protein Through Several Types of Proteins Bind to other Protein Through Several Types of InterfacesInterfaces

Protein FunctionProtein Function

Equilibrium Constant Describes Binding StrengthEquilibrium Constant Describes Binding Strength► Steady state or equilibrium: Steady state or equilibrium:

# association events/sec = # dissociation/sec# association events/sec = # dissociation/sec► From conc of two molecules and complex equilibrium constant can be From conc of two molecules and complex equilibrium constant can be

calculatedcalculated

Protein FunctionProtein FunctionEnzymes as CatalystsEnzymes as Catalysts► Make or break covalent bonds Make or break covalent bonds ► Speed up chemical reactions Speed up chemical reactions >> 10 106 6 foldfold

Stabilize transition stateStabilize transition state Decrease activation energyDecrease activation energy Increase local conc of substrate at catalytic siteIncrease local conc of substrate at catalytic site Hold reactants in proper orientation for chem rxnHold reactants in proper orientation for chem rxn Binding energy contributes directly to catalysisBinding energy contributes directly to catalysis

► Not consumed or changed during processNot consumed or changed during process

Protein FunctionProtein Function

Common Types of EnzymesCommon Types of EnzymesHydrolasesHydrolases IsomerasesIsomerases OxidoReductases OxidoReductases

Nuclease Nuclease PolymerasesPolymerases ATPasesATPases

ProteasesProteases KinasesKinases SynthasesSynthases

PhosphatasesPhosphatases

Protein FunctionProtein Function

Enzyme KineticsEnzyme Kinetics► VVmaxmax= how fast enzyme can process substrate, pt at which enzyme saturated = how fast enzyme can process substrate, pt at which enzyme saturated

w/substratew/substrate► Turnover Number= VTurnover Number= Vmaxmax/[enzyme]/[enzyme]

turnover ranges from 1-10,000 substrate molec/secturnover ranges from 1-10,000 substrate molec/sec► KKmm= substrate conc at V= substrate conc at Vmaxmax/2; measure of affinity /2; measure of affinity

Protein FunctionProtein FunctionLysozymeLysozyme► Natural antibiotic in egg white, tears, salivaNatural antibiotic in egg white, tears, saliva► Hydrolyzes polysaccharide chains residing in cell wall of Hydrolyzes polysaccharide chains residing in cell wall of

bacteriabacteria

Protein FunctionProtein Function

Specific Mechanism of Lysozyme HydrolysisSpecific Mechanism of Lysozyme Hydrolysis► Enzyme positions substrate bending critical chem bonds that participate in chem rxn Enzyme positions substrate bending critical chem bonds that participate in chem rxn ► Positions acidic side chain of Glu w/in active site to provide high conc of acidifying HPositions acidic side chain of Glu w/in active site to provide high conc of acidifying H++

ionsions► Negatively chged Asp stabilizes positive chged transition stateNegatively chged Asp stabilizes positive chged transition state

Protein FunctionProtein Function

General Mechanism for Enzyme ActivityGeneral Mechanism for Enzyme Activity► Active site contains atoms that speed up rxn Active site contains atoms that speed up rxn ► Substrate driven towards transition state upon binding to enzyme; Substrate driven towards transition state upon binding to enzyme;

shape of substrate chgs & critical bonds bentshape of substrate chgs & critical bonds bent► Covalent bond sometimes formed btwn substrate and side chain of Covalent bond sometimes formed btwn substrate and side chain of

enzymeenzyme► Restoration of side chain to original stateRestoration of side chain to original state

Protein FunctionProtein Function

Small Molecules Add Extra Functions to Small Molecules Add Extra Functions to ProteinsProteins

► Chromophores detect light; retinalChromophores detect light; retinal► Metal atoms assist w/ catalytic functions; Zn, Mg, FeMetal atoms assist w/ catalytic functions; Zn, Mg, Fe► Coenzymes (sm organic molec) provide functional grps; Coenzymes (sm organic molec) provide functional grps;

biotinbiotin

Protein FunctionProtein Function

Multienzyme ComplexesMultienzyme Complexes ► Increase the rate of cell metabolismIncrease the rate of cell metabolism► Product of enzyme A passed directly to enzyme B; product of enzyme B Product of enzyme A passed directly to enzyme B; product of enzyme B

passed to enzyme C; and so onpassed to enzyme C; and so on► Simulates intracellular membrane compartment; effectively increasing Simulates intracellular membrane compartment; effectively increasing

substrate conc at site of enzyme activitysubstrate conc at site of enzyme activity

Protein FunctionProtein Function

Regulation of Catalytic ActivityRegulation of Catalytic Activity► Negative FeedbackNegative Feedback► Positive RegulationPositive Regulation► AllosterismAllosterism

Protein FunctionProtein Function

AllosterismAllosterism

Protein FunctionProtein Function

Symmetric Protein Assemblies and Cooperative Allosterism:Symmetric Protein Assemblies and Cooperative Allosterism: sm chgs in ligand conc switches enzyme assembly from fully active to fully sm chgs in ligand conc switches enzyme assembly from fully active to fully inactive state via conformation changes that are transmitted across neighboring inactive state via conformation changes that are transmitted across neighboring subunitssubunits

Protein FunctionProtein Function

Allosteric Transition in Aspartate TranscarbamoylaseAllosteric Transition in Aspartate Transcarbamoylase► 6 catalytic subunits and 6 regulatory subunits6 catalytic subunits and 6 regulatory subunits► all or none transition between T-tense and R-relaxed stateall or none transition between T-tense and R-relaxed state► Active R state driven by binding of carbamoylphosphate and aspartateActive R state driven by binding of carbamoylphosphate and aspartate► Inactive T state driven by binding of CTP to regulatory dimersInactive T state driven by binding of CTP to regulatory dimers

Protein FunctionProtein Function

Regulation by Regulation by Phosphorylation/DephosphorylationPhosphorylation/Dephosphorylation

► Addition or removal of P grp carrying (2) negative chgs Addition or removal of P grp carrying (2) negative chgs can cause major conformation chg in proteincan cause major conformation chg in protein

► Phosphorylation/dephosphorylation of proteins= Phosphorylation/dephosphorylation of proteins= response to signals that specify chg in cell stateresponse to signals that specify chg in cell state

Protein FunctionProtein FunctionProtein KinaseProtein Kinase► transfers terminal P of ATP to OH grp of SER, Thr, or Tyrtransfers terminal P of ATP to OH grp of SER, Thr, or Tyr► 100’s ea specific for particular target100’s ea specific for particular target► Kinases share 250 aa catalytic domainKinases share 250 aa catalytic domain► Non-conserved aa flanking catalytic site or in loops w/in kinase domain confer specificityNon-conserved aa flanking catalytic site or in loops w/in kinase domain confer specificity

Protein FunctionProtein Function

Protein PhosphatasesProtein Phosphatases► Catalyzes the removal of P grpCatalyzes the removal of P grp► Some specific; some act on broad range of proteinsSome specific; some act on broad range of proteins

Protein FunctionProtein Function

Protein can Function as MicrochipProtein can Function as MicrochipCdk= cyclin dependent protein kinase Cdk= cyclin dependent protein kinase activity dependent upon 3 events:activity dependent upon 3 events:

1. binding of second protein cyclin1. binding of second protein cyclin2. phosphorylation of Thr side chain2. phosphorylation of Thr side chain3.3. dephosphorylation of Tyr side dephosphorylation of Tyr side chainchain

Cdk monitors specific set of cell Cdk monitors specific set of cell components acting as input-output components acting as input-output devicedevice

Protein FunctionProtein Function

GTP Binding ProteinsGTP Binding Proteins► Analogous to Proteins regulated by P/de-PAnalogous to Proteins regulated by P/de-P► Active when GTP bound; inactive when GTP hydrolyzedActive when GTP bound; inactive when GTP hydrolyzed

Protein FunctionProtein Function

Regulatory Proteins Control Activity of GTP Binding ProteinsRegulatory Proteins Control Activity of GTP Binding Proteins► GAP= GTPase activating protein; binds and induces hydrolysis GAP= GTPase activating protein; binds and induces hydrolysis ► GEF= Guanine nucleotide exchange factor; binds to GDP protein causing it to release GEF= Guanine nucleotide exchange factor; binds to GDP protein causing it to release

GDP in exchange for GAPGDP in exchange for GAP

Protein FunctionProtein Function

Large Protein Movements Generated from Small OnesLarge Protein Movements Generated from Small Ones► EF-Tu = elongation factor in protein synthesis, GTPaseEF-Tu = elongation factor in protein synthesis, GTPase

1.1. tRNA complexes w/ GTP bound form of EF-Tu w/ aa maskedtRNA complexes w/ GTP bound form of EF-Tu w/ aa masked

2.2. GTP hydrolysis occurs when tRNA binds to mRNA on ribosome; tRNA disassociates GTP hydrolysis occurs when tRNA binds to mRNA on ribosome; tRNA disassociates

3.3. GTP hydrolysis causes “Swtich helix” to swivel unmasking aaGTP hydrolysis causes “Swtich helix” to swivel unmasking aa

Protein FunctionProtein Function

Motor ProteinsMotor Proteins► Produce lg movements in cells such as:Produce lg movements in cells such as:

muscle contractionmuscle contraction

crawling and swimming of cellscrawling and swimming of cells

movement of chromosomesmovement of chromosomes

movement of organellesmovement of organelles

enzymes on DNAenzymes on DNA

► Possess moving parts as force generating machinesPossess moving parts as force generating machines

Protein FunctionProtein Function

ATP hydrolysis allows unidirectional series of conformational chgs to propel proteins ATP hydrolysis allows unidirectional series of conformational chgs to propel proteins along DNA along DNA

Protein FunctionProtein Function

Allosteric proteins harness energy derived from ATP Allosteric proteins harness energy derived from ATP hydrolysis, ion gradients, electron transport processes to hydrolysis, ion gradients, electron transport processes to pump ions or sm molecules across membranespump ions or sm molecules across membranes

CaCa2+ 2+ Pump of Sarcoplasmic ReticulumPump of Sarcoplasmic Reticulum

Protein FunctionProtein Function

Mechanism of CaMechanism of Ca2+2+ Pump Pump

Protein FunctionProtein Function

Structure of CaStructure of Ca2+ 2+ PumpPump► 10 transmembrane helices10 transmembrane helices► 4 transmembrane helices 4 transmembrane helices

provide Caprovide Ca2+2+ binding sites for binding sites for pumppump

► helices that bind Cahelices that bind Ca2+2+ wind wind around ea other forming cavity around ea other forming cavity btwn helices for Cabtwn helices for Ca2+2+

► ATP hydrolysis causes ATP hydrolysis causes conformation chgs that later conformation chgs that later cavity enabling Cacavity enabling Ca2+2+ to be to be pushed throughpushed through