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Chapter 3 Proteins:

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Weak Noncovalent Bonds/Interactions important to the folding of ... Binding energy contributes directly to catalysis. Not consumed or changed during process ... – PowerPoint PPT presentation

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Title: 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
6
Protein Shape and Structure
Weak Noncovalent Bonds/Interactions important to
the folding of polypeptide chain
7
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

9
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
10
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
11
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

12
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

13
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
16
Protein Shape and Structure
  • Larger proteins often contain more than one
    polypeptide
  • Proteins can serve as subunits for assembly of
    large structures
  • Self Assembly

17
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

18
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

19
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

20
Protein Function
  • Proteins Bind to other Protein Through Several
    Types of Interfaces

21
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

24
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

26
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

27
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

28
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

29
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

30
Protein Function
  • Regulation of Catalytic Activity
  • Negative Feedback
  • Positive Regulation
  • Allosterism

31
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

33
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

36
Protein Function
  • Protein Phosphatases
  • Catalyzes the removal of P grp
  • Some specific some act on broad range of proteins

37
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

39
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

44
Protein Function
  • Mechanism of Ca2 Pump

45
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
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