Chapter 3 Proteins:

1
Chapter 3 Proteins

• Shape, Structure, and Function

2
Proteins Execute Cell Functions

• Enzymes
• Channels and pumps
• Signal Molecules
• Messengers
• Molecular Machines
• Structural Support
• Cell Recognition

3
Protein Shape and Structure
Peptide Bond Links Amino Acids into Polypeptide
Chain
4
Protein Shape and Structure

• Evolution fine-tuned structure and chemistry
• Shape dictated by amino acid sequence
• polypeptide backbone
• side chains

5
Protein Shape and Structure
Sequence Determines Structure
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Protein Shape and Structure
Weak Noncovalent Bonds/Interactions important to
the folding of polypeptide chain
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Protein Shape and Structure

• Fold into Conformation of Lowest Energy
• Common Folding Patterns
• alpha helix
• Beta Sheet
• Coiled Coils

8
Protein Shape and Structure

• Levels of organization protein structure
• primary aa seqeunce
• secondary stretches of alpha helix, beta sheets
• tertiary3d organization
• quartenarycomplete structure of protein w/ gt 1
  poly-peptide chain

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Protein Shape and Structure

• Protein Domain Fundamental Unit of Organization
• independently folding unit
• 40-350 aa modular unit combine to form larger
  proteins
• different domains have different functions
• Fold central core of domain comprised of beta
  sheets and alpha helices in various combinations
  limited number

Short signature sequences identify homologous
protein domains
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Protein Shape and Structure
Domain shuffling during the course of evolution
Percentage of total genes in respective genomes
containing one or more copies of a particular
protein domain
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Protein Shape and Structure

• Protein Module
• Smaller than an average domain, generally 40-200
  aa
• Particular versatile structures
• Easily integrated into other proteins form parts
  of many different proteins

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Protein Shape and Structure

• Protein Families Evolved
• similar 3d structure
• portions or aa sequence conserved
• non-conserved portions impart new functionality
• serine proteases
• homeodomain proteins
• kinases
• immunoglobulins

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Protein Shape and Structure

• Sequence Homology Searches
• Amino Acids Sequence Threading
• Modules form parts of many different proteins

14
Protein Shape and Structure
15
Protein Shape and Structure
Larger proteins can assemble from identical
monomeric subunits
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Protein Shape and Structure

• Larger proteins often contain more than one
  polypeptide
• Proteins can serve as subunits for assembly of
  large structures
• Self Assembly

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Protein Function

• Function of protein dictated by physical
  interactions w/ other molecules
• specificity and ligand affinity governed by
  multiple weak noncovalent bonds
• active/binding site often cavity on protein
  surface formed by neighboring aa or aa that may
  belong to different portions of polypeptide

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Protein Function

• Conformation determines chemistry
• Regions adjacent to active or ligand binding site
  may restrict water to increase ligand binding
• Clustering of polar or chged residues can alter
  chemical reactivity
• Type and orientation of exposed aa side chains
  govern chemical reactivity

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Protein Function

• Evolutionary tracing to determine sites
  critical to protein function
• 3d structure of protein family members are
  similar even when aa homology falls to 25
• Map unchg aa or nearly unchg from all known
  family members onto 3d structure of one family
  member
• Most invariant positions often on surface and
  represent ligand binding site

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Protein Function

• Proteins Bind to other Protein Through Several
  Types of Interfaces

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Protein Function

• Equilibrium Constant Describes Binding Strength
• Steady state or equilibrium
• association events/sec dissociation/sec
• From conc of two molecules and complex
  equilibrium constant can be calculated

22
Protein Function

• Enzymes as Catalysts
• Make or break covalent bonds
• Speed up chemical reactions gt 106 fold
• Stabilize transition state
• Decrease activation energy
• Increase local conc of substrate at catalytic
  site
• Hold reactants in proper orientation for chem rxn
• Binding energy contributes directly to catalysis
• Not consumed or changed during process

23
Protein Function

• Common Types of Enzymes
• Hydrolases Isomerases OxidoReductases
• Nuclease Polymerases ATPases
• Proteases Kinases Synthases
• Phosphatases

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Protein Function

• Enzyme Kinetics
• Vmax how fast enzyme can process substrate, pt
  at which enzyme saturated w/substrate
• Turnover Number Vmax/enzyme
• turnover ranges from 1-10,000 substrate
  molec/sec
• Km substrate conc at Vmax/2 measure of affinity

25
Protein Function

• Lysozyme
• Natural antibiotic in egg white, tears, saliva
• Hydrolyzes polysaccharide chains residing in cell
  wall of bacteria

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Protein Function

• Specific Mechanism of Lysozyme Hydrolysis
• 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 H ions
• Negatively chged Asp stabilizes positive chged
  transition state

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Protein Function

• General Mechanism for Enzyme Activity
• Active site contains atoms that speed up rxn
• Substrate driven towards transition state upon
  binding to enzyme shape of substrate chgs
  critical bonds bent
• Covalent bond sometimes formed btwn substrate and
  side chain of enzyme
• Restoration of side chain to original state

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Protein Function

• Small Molecules Add Extra Functions to Proteins
• Chromophores detect light retinal
• Metal atoms assist w/ catalytic functions Zn,
  Mg, Fe
• Coenzymes (sm organic molec) provide functional
  grps biotin

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Protein Function

• Multienzyme Complexes
• Increase the rate of cell metabolism
• Product of enzyme A passed directly to enzyme B
  product of enzyme B passed to enzyme C and so on
• Simulates intracellular membrane compartment
  effectively increasing substrate conc at site of
  enzyme activity

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Protein Function

• Regulation of Catalytic Activity
• Negative Feedback
• Positive Regulation
• Allosterism

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Protein Function

• Allosterism

32
Protein Function

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

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Protein Function

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

34
Protein Function

• Regulation by Phosphorylation/Dephosphorylation
• Addition or removal of P grp carrying (2)
  negative chgs can cause major conformation chg in
  protein
• Phosphorylation/dephosphorylation of proteins
  response to signals that specify chg in cell state

35
Protein Function

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

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Protein Function

• Protein Phosphatases
• Catalyzes the removal of P grp
• Some specific some act on broad range of proteins

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Protein Function

• Protein can Function as Microchip
• Cdk cyclin dependent protein kinase activity
  dependent upon 3 events
• 1. binding of second protein cyclin
• 2. phosphorylation of Thr side chain
• 3. dephosphorylation of Tyr side chain
• Cdk monitors specific set of cell components
  acting as input-output device

38
Protein Function

• GTP Binding Proteins
• Analogous to Proteins regulated by P/de-P
• Active when GTP bound inactive when GTP
  hydrolyzed

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Protein Function

• Regulatory Proteins Control Activity of GTP
  Binding Proteins
• GAP GTPase activating protein binds and induces
  hydrolysis
• GEF Guanine nucleotide exchange factor binds to
  GDP protein causing it to release GDP in exchange
  for GAP

40
Protein Function

• Large Protein Movements Generated from Small Ones
• EF-Tu elongation factor in protein synthesis,
  GTPase
• 1. tRNA complexes w/ GTP bound form of EF-Tu w/
  aa masked
• 2. GTP hydrolysis occurs when tRNA binds to mRNA
  on ribosome tRNA disassociates
• 3. GTP hydrolysis causes Swtich helix to
  swivel unmasking aa

41
Protein Function

• Motor Proteins
• Produce lg movements in cells such as
• muscle contraction
• crawling and swimming of cells
• movement of chromosomes
• movement of organelles
• enzymes on DNA
• Possess moving parts as force generating machines

42
Protein Function

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

43
Protein Function

• Allosteric proteins harness energy derived from
  ATP hydrolysis, ion gradients, electron transport
  processes to pump ions or sm molecules across
  membranes
• Ca2 Pump of Sarcoplasmic Reticulum

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Protein Function

• Mechanism of Ca2 Pump

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Protein Function

• Structure of Ca2 Pump
• 10 transmembrane helices
• 4 transmembrane helices provide Ca2 binding
  sites for pump
• helices that bind Ca2 wind around ea other
  forming cavity btwn helices for Ca2
• ATP hydrolysis causes conformation chgs that
  later cavity enabling Ca2 to be pushed through