Proteins

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Proteins

• AHMP 5406

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Objectives

• Discuss the shape and structure of proteins.
• List some different functions of proteins.
• Describe the process of protein binding to
  another molecule.
• Discuss the class of proteins called enzymes and
  their catalytic functions.
• Explain the different ways in which the catalytic
  activities of enzymes are regulated.
• Discuss the function of proteins as cellular
  regulators.
• Discuss the function of motor proteins and
  membrane-bound transporter proteins.

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Structure of Proteins

• Def a long chain of amino acids linked by
  covalent peptide bonds
• AKA polypeptides
• Shape is determined by amino acid sequence
• Polypeptide backbone forms a repetitive core
• Characteristics of a protein are due to their
  amino acid side chains

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Amino acid structure
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AA side chain characteristics

• Can be
• Polar
• Negatively charged - Acidic
• Positively charged - Basic
• Uncharged but still polar
• Nonpolar
• hydrophobic

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Shape of Proteins

• The final folded structure of a protein is
  referred to as its conformation
• Unfolded proteins are denatured
• Fold according to lowest energy
• Three types of bonds determine protein
  conformation
• Ionic bonds
• Hydrogen bonds
• Van der Waals
• attractions

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Interactions in aqueous environments

• Polar side chains interact with water
• Nonpolar (hydrophobic) side chains interact with
  each other

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Hydrogen bonds

• Help stabilize protein conformation

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Common Folding Patterns

• a helix
• Cell membrane proteins
• Formed when polypeptide chains twist around
  themselves
• b sheet
• Formed when chains run parallel to each other

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Organizational Units

• Four structural levels
• Primary AA sequence
• Secondary a helices or b sheets
• Tertiary 3D shape of pp chain
• Quaternary complex of more than one pp chain
• Protein domain is the fundamental unit
• Substructure that can fold independently into a
  compact and stable structure

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Selection and protein structure

• Few of large of polypeptide chains will be
  useful
• Typical protein is approx. 300 AA in length
• Given 20 AA we can have 20300 possible
  polypeptide chains
• Only a small fraction would be stable
• Natural selection eliminates proteins that are
  unstable and have unpredictable biochemical
  properties

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Protein Families

• Evolutionarily successful proteins can be
  modified or duplicated
• Allows for novel functions
• Structurally related proteins can be classified
  into protein families
• E.g. serine proteases include
• Chymotrypsin, trypsin, elastase and blood
  clotting proteases
• Protein families are recognized by DNA sequence
  similarities

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Domain Shuffling

• Multidomain proteins most likely originated from
  the joining of DNA sequences that code for each
  domain
• Referred to as domain shuffling
• Allow for novel combinations and functions
• Particularly mobile domains are called protein
  modules

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Protein Modules

• Relatively small, 40-200 AA
• Have versatile structures
• Can be easily integrated into other proteins

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Large proteins contain multiple polypeptide chains

• Weak noncovalent bonds allow proteins to bind to
  each other
• Binding site is area of interaction between
  protein and another molecule (or protein)
• Thus protein subunits can form larger proteins

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Protein structures and uses

• Some proteins from long helical filaments
• Identical protein subunits have complementary
  binding sites on either end
• Depending on position of binding site can also
  form rings
• Actin found in the cytoskeleton

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Protein structures and uses

• Fibrous protein molecules
• Elongated three-dimensional structures
• Used for spanning relatively long distances in
  the cell
• a-keratin formed by two a-helices
• Coiled coil
• Intermediate filaments
• Cytoskeleton
• Extracellular matrix of cells
• Collagen
• Triple helix
• Elastin found in resilient tissues

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Extracellular proteins

• Stabilized by covalent cross-linkages
• Bind two amino acids
• Connect different polypeptide chains
• Most common are disulfide bonds
• Formed as proteins are being modified for export
• Endoplasmic reticulum

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Proteins can be part of supramolecular structures

• Same binding mechanisms can allow proteins to
  form larger structures
• E.g. enzyme complexes, ribosomes, protein
  filaments, viruses, and membranes
• Multiple protein contacts adds stability to
  overall structure

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Large protein assembly

• Assembly on a core
• a protein core of macromolecule provides
  scaffold
• protein length determined by the core length
• TMV tail length determined by RNA chain
• Thin filaments in muscle

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Large protein assembly

• Accumulated Strain
• Assembly is terminated due to strain of polymer
• Addition of subunits becomes energetically
  unfavorable
• Vernier Mechanism
• Vernier scale
• Molecules based on differently sized subunits
  grow until their ends match

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Protein Function
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Intro to protein binding

• Biological properties of proteins depends on
  interaction with other molecules
• Binding is usually very specific
• A ligand is a substance that binds to a protein
  by noncovalent bonds
• Ion
• Small molecule
• Macromolecule
• Ligand binds to binding site

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Protein conformation chemical reactivity

• Binding site modification
• Folding can create specific binding sites
• Ex. Water can compete with ligand
• Clustering nonpolar AAs will repel water
• Or activate nonreactive AAs

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Protein conformation chemical reactivity

• Reactive amino acids at binding site
• Ex. If you put many neg side chains together
• Then you increase affinity for positively charged
  ligand
• Can change reactivity of unreactive side group

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Types of binding interfaces

• Surface-string
• Protein contacts extended loop of another
  proteins polypeptide chain
• Ex. SH2 domain can recognize phosphorylated
  polypeptide loop on other proteins
• Helix-Helix
• AKA coiled-coil
• Surface-surface
• Most common
• Tight interactions because many weak bonds
• Very specific

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Antibody binding sites

• Antibodies proteins produced by immune system in
  response to foreign molecules
• Antibodies bind to a target called antigen
• Have two identical binding sites (Y)
• Formed by many loops
• Which are good for binding
• B/C many chemical groups can surround the ligand

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Enzymes

• Make and break covalent bonds
• Bind to ligands called substrates
• Convert substrates into products
• Can also speed up reactions catalysts
• Enzymes are grouped into classes by type of
  reaction

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Enzyme Classes

• Hydrolases hydrolytic cleavage
• Nucleases break nucleotide bonds
• Proteases break amino acid bonds
• Phosphatases remove phosphate groups
• ATPases hydrolyze ATP
• Synthases synthesize molecules by anabolic
  reactions
• Isomerases rearrange bonds within molecules
• Polymerases synthesize DNA and RNA
• Kinases add phosphate groups to molecules
• Oxido-Reductases one molecule is oxidized
  another is reduced

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Substrate binding

• First step in enzyme catalysis
• E enzyme
• S substrate
• P product
• Enzymes can be reused

E S ? ES ? EP ? E P
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Enzymes can speed up reactions

• By stabilizing transition states of substrates
• Concentrating substrate molecules at binding
  sites
• Binding energy contributes to catalysis
• Decreases activation energy

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Example of rate accelerations
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Small molecules add functionality

• Non protein molecules can allow difficult
  reactions to occur
• Hemoglobin
• Four heme groups and iron atom
• Carboxypeptidase
• Cuts polypeptide chains
• Has zinc in its active site to assist reaction
• Coenzymes organic molecules
• Vitamins can not be produced by cell

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Regulation of catalytic activities

• Expression of gene
• Compartmentalization
• Targeted degradation of enzymes
• Another molecule binds to a regulatory site
• Affects rate of reaction
• Feedback inhibition
• Enzyme early in the reaction is affected by later
  product
• AKA negative regulation
• Positive regulation
• Later product reinforces enzyme activity

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Allosteric enzymes

• Have two binding sites
• Active site
• Regulatory site
• Binding of regulatory site can lead to
  conformational changes
• Affects rate of reaction

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Enzyme Linkage

• One ligand can affect binding of another ligand
• Due to conformation change
• These two sites are coupled
• Can increase or decrease affinities for ligands.

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Phosphorylation

• Regulates enzyme function
• Causes conformational changes
• Phosphate can contribute to binding site
• Phosphorylation of enzymes can be signaled
• External stimulus
• Protein kinases

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Protein kinases and phosphatases

• Proteins are phosphorylated by addition of
  terminal P group from ATP
• Serine, Threonine or Tyrosine
• Reaction catalyzed by protein kinase
• Unidirectional due to energy release

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Protein kinases and phosphatases

• Protein phosphatases
• Dephosphorylate proteins
• Remove phosphate group

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Proteins as cellular regulators

• Signaling devices
• Cdk
• controls cell cycles
• only active if bound to cyclin and phosphate
• and if also simultaneously dephosphorylated
  somewhere else
• Src another example
• signal intergrators

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Proteins as cellular regulators
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GTPases

• Bound GTP is hydrolyzed
• Causes conformational change
• Activates protein
• Controlled by regulatory proteins
• GAP GTPase activating protein
• GEF Guanine nucleotide exchange factor

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Elongation Factors

• Some proteins required as assembly factors
• E.g. Building polymers
• EF-Tu protein
• Elongation factor in protein synthesis
• Loads each amino-acyl tRNA molecule onto ribosome
• tRNA bound and masked by EF-TU when GTP is
  present
• If bound to ribosome and phosphate group is
  hydrolyzed (GTP ? GDP)
• tRNA is unmasked
• tRNA transfers AA to the polypeptide

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Motor Proteins

• Function to move other proteins
• Muscle contractions
• Chromosome movement
• Organellar movement
• Work by unidirectional conformation changes
• By coupling a change with ATP hydrolysis
• Energetically unfavorable to go in reverse

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Membrane Transport Proteins

• Harness energy
• ATP hydrolysis
• Ion gradients
• Electron transport processes
• Ex. Ca2 pump
• Muscle cells
• Specialized organelle sarcoplasmic reticulum
• Pumps Ca ions out of cell to maintain low levels
• ATP hydrolysis drives conformation change

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