Protein Structure and Function

Chapter 3
Protein Structure and Function
Broad functional classes
So Proteins have structure and
function... Fine!
-Why do we care to know
more????
Understanding functional
architechture gives us POWER
to:
•Diagnose and find reasons for
diseases
•Create modifying drugs
•Engineer our own designerproteins
Protein structure determines function
DNA
(mRNA)
Translation:
Translation into 3D structure:
Modifications:
Chemical modification of aminoacids
Interaction with other molecules
Proteolytic cleavage
(Location)
3D structure determines function:
New 3D structure
New function
a
a
a
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Proteins are single, unbranched chains of amino acid monomers
There are 20 different amino acids
The amino acid sidechains in a peptide can become modified, extending the functional repetoire of aminoacids to more
than hundred different amino acids.
A protein’s amino acid sequence determines its three-dimensional structure (conformation)
In turn, a protein’s structure determines the function of that protein
Conformation (=function) is dynamically regulated in several different ways
All amino acids have the same general structure
but the side chain (R group) of each is different
Cα
R:
Hydrophilic:
Basic
Acidic
Non-charged
Hydrophobic
“Special”
Hydrophilic amino acids
Hydrophobic
and “special” amino acids
Peptide bonds connect amino acids into linear
chains
Backbone
Side-chains
Side chain modifications change the chemical
(functional) properties of proteins
Acetylation
Phosphorylation
Hydroxylation
Methylation
Carboxylation
Glycosylation
Ubiquitylation
=> Expanding the
repetoire of existing
amino acid side-chains to
> 100 variations!
Four levels of structure determine the shape of proteins
a Primary: the linear sequence of
amino acids
peptide bonds
a Secondary: the localized
organization of parts of a
polypeptide chain (e.g., the
α helix or β sheet)
backbone hydrogen bonds
a Tertiary: the overall, threedimensional arrangement of the
polypeptide chain
hydrophobic interactions, hydrogen
bonds (non-covalent bonds in
general) and sulfur-bridges
a Quaternary: the association of two
or more polypeptides into a multisubunit complex
Primary and secondary structure
(example: hemagglutinin)
β-strand
α-helix
Secondary structure
α Helix
β Sheet
β (U)-turn
Tertiary structure
Motifs are regular combinations of secondary structures. Motifs form domains!
Three examples of Motifs from different types of DNA-binding proteins
Tertiary structure
Structural, functional or topological domains are modules of secondary and tertiary
structure
Each of these proteins
contain the EGF
globular domain.
- But each of these
proteins have a different
function
Globular domain
Tertiary structure
Different graphical representations of the same protein
(tertiary structure)
Quaternary structure
Multiprotein complexes: molecular machines
Sequence homology suggests functional and
evolutionary relationships between proteins
When the stucture of a newly discovered protein is known, comparison to other proteins
across species can help predict function
Folding, modification, and degradation of proteins
The life of a protein can briefly be described as: synthesis,
folding, modification, function, degradation.
a A newly synthesized polypeptide chain must undergo folding
and often chemical modification to generate the final protein
a All molecules of any protein species adopt a single
conformation (the native state), which is the most stably
folded form of the molecule
a Most proteins have a limited lifespan before they are
degraded (turn-over time)
Aberrantly folded proteins are implicated in slowly
developing diseases
An amyloid plaque in
Alzheimer’s disease is
a tangle of protein
filaments
The information for protein folding is encoded in the
sequence
Folding of proteins in vivo is promoted by chaperones
Large 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 protein
Folding of proteins in vivo is promoted by chaperones
Large proteins with a lot of secondary structure may require assisted folding to avoid
aggregation of unfolded protein
- Chaperones and chaperonins prevent aggregation of unfolded protein
Functional design of proteins
a Protein function often involves conformational changes
a Proteins are designed to bind a range of molecules (ligands)
`Binding is characterized by two properties: affinity and specificity
a Antibodies and enzymes exhibit precise ligand/substratebinding specificity
But can have variable affinities
a Enzymes are highly efficient and specific catalysts
`An enzyme’s active site binds substrates(ligands) and carries out
catalysis
Antibody/antigen interaction: an example for ligand-binding with
high affinity and specificity
Enzymes have high substrate affinity sites and
catalytic sites
Kinetics of an enzymatic reaction are described by
Vmax and Km
Kinetics of an enzymatic reaction are described by Vmax
and Km
Enzymes in one pathway can be physically associated
Mechanisms that regulate protein activity
a Altering protein synthesis rate and proteasomal degradation
a Allosteric transitions
` Release of catalytic subunits, active Ù inactive states, cooperative binding
of ligands
a Chemical modification:
` Phosphorylation, acetylation etc. Ù dephosphorylation, deacetylation etc.
a Proteolytic activation
a Compartmentalization
Protein degradation via the ubiquitin-mediated pathway
ATP
Cells contain several other pathways for
protein degradation in addition to this
pathway
Allosteric transitions: Cooperative binding of ligands
Sigmoidal curve indicates cooperative binding (of
ligands, substrates, ca ions) in contrast to standard
Michaelis-Menten Kinetics
Conformational changes induced by Ca2+ binding to
calmodulin
Cooperative binding of
calcium: binding of one
calcium enhances the
affinity for the next
calcium
When 4 calcium are
bound a major allosteric
conformational change
occurs
Calmodulin is a switch
protein because this
effect in turn regulates
other proteins bound by
the compact calmodulin
Another class of switch proteins: GTPases
Chemical modification
Example: Phosphorylation Ùdephosphorylation
Proteolytic cleavage of proinsulin to produce active insulin
Compartmentalization
Example:Membrane proteins
a Each cell membrane has a set of specific
membrane proteins that allows the
membrane to carry out its activities
a Membrane proteins are either integral
or peripheral
a Integral transmembrane proteins contain
one or more transmembrane α helices
a Peripheral proteins are associated with
membranes through interactions with
integral proteins
Schematic of membrane proteins in a lipid bilayer
Mechanisms that regulate protein activity
a Altering protein synthesis rate and proteasomal degradation
a Allosteric transitions
` Release of catalytic subunits, active Ù inactive states, cooperative binding
of ligands
a Chemical modification:
` Phosphorylation, acetylation etc. Ù dephosphorylation, deacetylation etc.
a Proteolytic activation
a Compartmentalization
Example containing all levels of regulatin of protein activity
GFP-tagged GLUT4
Now that you KNOW the basic principles of protein structure and
function you can UNDERSTAND:
Protein and Proteome
Analytical techniques
Purifying, detecting, and characterizing proteins
a A protein must be purified to determine its structure and
mechanism of action
a Detecting known proteins can be usefull for diagnostic
purposes
a Molecules, including proteins, can be separated from other
molecules based on differences in physical and chemical
properties (size, mass, density, polarity, affinity...)
`Elementary toolbox includes: centrifugation, electrophoresis, liquid
chromatography (LC), spectrometry, ionization/radiation.
-applied in various advanced forms and combinations.
Centrifugation can separate molecules that
differ in mass or density
Electrophoresis separates molecules
according to their charge:mass ratio
SDS-polyacrylamide
gel electrophoresis
Even coating of proteins
allows even charge
distribution -> larger mass
= higher total charge
Two-dimensional electrophoresis separates molecules
according to their charge and their mass
Highly specific enzymes and antibody assays can
detect individual proteins
Immunoblot (= Western Blot)
based on affinity
Liquid chromotography (LC):
Separation of proteins by size: gel filtration chromatography
Add mobile phase:
buffer
Stationary phase:
Separation of proteins by charge: ion exchange
chromatography
Also: Reversed-phase LC: separation by hydrophobicity
Stationary phase: non-polar,
Mobile phase: moderately polar
Separation of proteins by specific binding to another
molecule: affinity chromatography
Proteomics, the analysis of complex protein
mixtures
a Genome databases allow prediction of genes -> protein
primary structure
a Each protein can be fragmented into peptides which are
composed of aa’s.
a Each aa has a unique mass to charge ratio at a given pH
a Each protein therefore has a unique peptide-fingerprint
a Technique: proteins->peptides->mass/charge ratio
measurement -> compare against whole proteome (genome
based) database -> identify proteins
Time-of-flight mass spectrometry measures the
mass of proteins and peptides
Matrix-Assisted-Laser-Desorption/Ionization Time-offlight mass spectrometry (MALDI-TOF MS)
MS spectrum
Example of a proteome analysis workflow
Cell/tissue of interest
Isolate organelles
(fractionation)
Confirm organelle-specific
proteins
Subfractionate, detect
peptides, identify
corresponding proteins
X-ray crystallography is used to determine protein
structure
Other techniques such as cryoelectron microscopy and NMR
spectroscopy may be used to solve the structures of certain types of
proteins