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 POWERto:•Diagnose and find reasons for diseases•Create modifying drugs•Engineer our own designer-proteins

DNA

(mRNA)

Translation:

Translation into 3D structure:

3D structure determines function:

Modifications:Chemical modification of aminoacids

Interaction with other molecules

Proteolytic cleavage

(Location)

New 3D structure

New function

Proteins are single, unbranched chains of amino acid monomersThere are 20 different amino acidsThe 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 proteinConformation (=function) is dynamically regulated in several different ways

Protein structure determines function

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

Backbone

Side-chains

Peptide bonds connect amino acids into linear chains

Side chain modifications change the chemical (functional) properties of proteins

Glycosylation

Ubiquitylation

=> Expanding the repetoire of existing amino acid side-chains to > 100 variations!

Acetylation

Phosphorylation

Hydroxylation

Methylation

Carboxylation

Four levels of structure determine the shape of proteins

Primary: the linear sequence of amino acids peptide bonds

Secondary: the localized organization of parts of a polypeptide chain (e.g., the α helix or β sheet)backbone hydrogen bonds

Tertiary: the overall, three-dimensional arrangement of the polypeptide chainhydrophobic interactions, hydrogen bonds (non-covalent bonds in general) and sulfur-bridges

Quaternary: the association of two or more polypeptides into a multi-subunit complex

Primary and secondary structure (example: hemagglutinin)

β-strand α-helix

Secondary structure

α Helixβ Sheet

β (U)-turn

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

Globular domain

Tertiary structure

Each of these proteins contain the EGF globular domain.

- But each of these proteins have a different function

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 newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final protein

All molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the molecule

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

Protein function often involves conformational changes

Proteins are designed to bind a range of molecules (ligands)Binding is characterized by two properties: affinity and specificity

Antibodies and enzymes exhibit precise ligand/substrate-binding specificityBut can have variable affinities

Enzymes are highly efficient and specific catalystsAn 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 Vmaxand Km

Enzymes in one pathway can be physically associated

Mechanisms that regulate protein activity

Altering protein synthesis rate and proteasomal degradation

Allosteric transitionsRelease of catalytic subunits, active inactive states, cooperative binding of ligands

Chemical modification: Phosphorylation, acetylation etc. dephosphorylation, deacetylation etc.

Proteolytic activation

Compartmentalization

Protein degradation via the ubiquitin-mediated pathway

Cells contain several other pathways for protein degradation in addition to this pathway

ATP

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 allostericconformational changeoccurs

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 dephosphorylation

Proteolytic cleavage of proinsulin to produce active insulin

CompartmentalizationExample:Membrane proteins

Each cell membrane has a set of specificmembrane proteins that allows themembrane to carry out its activitiesMembrane proteins are either integralor peripheralIntegral transmembrane proteins containone or more transmembrane α helicesPeripheral proteins are associated withmembranes through interactions withintegral proteins

Schematic of membrane proteins in a lipid bilayer

Mechanisms that regulate protein activity

Altering protein synthesis rate and proteasomal degradation

Allosteric transitionsRelease of catalytic subunits, active inactive states, cooperative binding of ligands

Chemical modification: Phosphorylation, acetylation etc. dephosphorylation, deacetylation etc.

Proteolytic activation

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 ProteomeAnalytical techniques

Purifying, detecting, and characterizing proteins

A protein must be purified to determine its structure and mechanism of action

Detecting known proteins can be usefull for diagnostic purposes

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-polyacrylamidegel 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 chromatographyAdd mobile phase: buffer

Stationary phase:

Separation of proteins by charge: ion exchange chromatography

Also: Reversed-phase LC: separation by hydrophobicityStationary 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

Genome databases allow prediction of genes -> protein primary structureEach protein can be fragmented into peptides which are composed of aa’s.Each aa has a unique mass to charge ratio at a given pHEach protein therefore has a unique peptide-fingerprint

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-of-flight 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