Download Three-Dimensional Structure of Proteins

Three-Dimensional Structure of Proteins
Rotation around the a-Carbon in a
Polypeptide
A Sterically Nonallowed Conformation
The a Helix and b Pleated Sheet
Conformationally
allowable structures
where backbone is
optimally Hbonded (linear Hbonds).
a Helix (3.613 Helix):
•3.6 residues/turn
•Rise = 0.15 nm/
residue
•13-atom hydrogenbonded loop
Linus Pauling and Robert
Corey, 1950
b Pleated Sheet:
•Anti-parallel or parallel
•2.0 residues/”turn”
•0.34 nm/residue (antiparallel) or 0.32
nm/residue (parallel)
Linus Pauling and Robert
Corey, 1951
Antiparallel and Parallel b Pleated Sheets
Other Secondary Structures
310 Helix:
•3 residues/turn
•0.20 nm/residue
•10-atom hydrogenbonded loop
 Helix (4.416 Helix):
•4.4 residues/turn
•0.12 nm/residue
•16-atom hydrogenbonded loop
Idealized Helices
Hydrogen Bonding Patterns for Different
Helices
Ramachandran Plot
G.N. Ramachandran, 1963
Fibrous Proteins*
Proteins with an elongated or filamentous form, often dominated by
a single type of secondary structure over a large distance.
Most fibrous proteins are associated with connective
tissue and help provide mechanical strength to the
tissue.
*vs.
globular proteins
Structure of Keratin and Keratin-Type
Intermediate Filaments
Keratin is a principal
component of hair,
horn, nails and
feathers.
Adjacent polypeptide
chains also crosslinked by
disulfide bonds.
Disulfide bond patterns
between are what
determine whether human
hair is straight or curly.
Coiled-Coil a-Helical Dimer of a Keratin
Amphipathic a helices:
Residues a, d, a’ and d’
hydrophobic, other residues
hydrophilic.
Structure of Silk Fibroin
Silk made by
silkworms and
spiders.
Composed of
microcrystalline array
of antiparallel b pleated
sheets where each b
strand has alternating
Gly and Ala or Ser
residues.
Structure of Collagen Fibers
Collagen is the most
abundant vertebrate
protein and the
major stress-bearing
component of
connective tissue
(bone, teeth,
cartilage, tendon)
and fibrous matrix of
skin and blood
vessels.
•3 intertwined lefthanded helices
•3.3 residues/turn
•Repeating Gly-X-Y
(X often Pro, Y often
Pro or hydroxyPro)
The Collagen Triple Helix (Tropocollagen)
Interactions between strands
G.N. Ramachandran, 1955
Tropocollagen with Gly Ala
substitution (yellow)
Post-Translational Modifications in
Collagen
Collagen contains unusual, oxidized and crosslinked
lysine residues.
Lysyl oxidase is the enzyme that oxidizes lysine
residues to the aldehyde allysine, which then forms
the crosslinks.
Hydroxyproline is also found in collagen. (Some lysine
residues also hydroxylated.) The enzyme required for
hydroxylation of proline residues is prolyl hydroxylase, a
vitamin C-dependent enzyme.
Scurvy is caused by reduced hydroxyproline in collagen as a
result of vitamin C deficiency.
Biosynthesis and Assembly of Collagen
Globular Proteins
Proteins with a compact folded structure (with an interior and
exterior), generally containing different types of secondary structure
elements as well as irregular regions.
The vast majority of proteins are globular.
Ribbon Diagram Showing Secondary
Structures in a Globular Protein
Some Globular Protein Structures
Myoglobin
Triose phosphate isomerase (complex of 2 subunits)
Hemoglobin
(complex of 4
polypeptide
chains or
subunits)
20S Proteasome (complex of 28 subunits)
Additional Elements of Structure: Turns
R2 often Pro
R3 never Pro
Most common
type of turn
R2 often Pro
R3 never Pro
b turns
g turn
trans-Pro (above) or cis-Pro (in Type VI b turns) often found in turns.
Turns with cis-Proline: Type VI b turns
Type VIa b turn
Type VIb b turn
cis-trans Isomerization of Proline
Residues
Peptidyl-prolyl cis-trans isomerases (rotamases) accelerate the isomerization.
Additional Elements of Structure: Loops
•Irregularly structured elements
•More disordered and flexible than turns
•Connects secondary structure elements
•Variable in length and shape
•Frequently form binding sites and enzyme active sites
The N- and C-terminal arms of proteins are also generally more disordered and irregularly structured.
Domain and Motifs in Globular Proteins:
Supersecondary Structure
Some Common Motifs Found in Proteins
b hairpin
bab motif
aa motif
b barrels
ab barrel
Solving 3-D Structure of Proteins
•X-ray crystallography
–Must crystallize protein.
–Structure solved is of protein packed in crystal
and not protein in solution; however, protein
crystals usually have high water content (and so
solution-like in structure).
–Almost no limit to size of protein.
•Nuclear magnetic resonance (NMR)
–Solution structure.
–Can be used to look at dynamics.
–Only possible at this time for proteins with a Mr of
< ~50,000.
X-Ray Crystallography Used to Solve First 3-D
Protein Structures
Max Perutz with model
of hemoglobin and John
Kendrew with model of
myoglobin in 1962
X-Ray Crystallography to Solve Protein
Structures
Protein crystals
3-D
structure of
protein
X-ray
diffraction
pattern
Electron
density map
Nuclear Magnetic Resonance (NMR)
Comparison of X-Ray and NMR Structures of
Bovine Pancreatic Trypsin Inhibitor
Both X-ray crystallography and NMR
methods usually yield very similar
structures.
Crystallized proteins generally adopt a
conformation very similar to the
“averaged” NMR solution structure.
Molecular Motion in Proteins
Proteins are not static structures but are highly dynamic:
•“Breathing” - molecular-scale vibrations and oscillations
•Larger-scale conformational changes in both secondary
structural elements and whole domains
Looking at Protein Dynamics
•NMR or lower-resolution methods (e.g., circular
dichroism, absorption spectroscopy, fluorescence
spectroscopy, hydrogen exchange MS) are useful for
looking at dynamics in solution.
•X-ray crystallography is not suited for looking at
dynamics, despite being the most powerful method for
determining basic structure. However, if you can make
separate crystals with protein in different conformations
(e.g., protein bound to ligand or substrate vs. unbound
protein), you can compare structures of both states.
Protein Folding and Stability
Thermodynamics of Folding: Factors to
Consider
• Conformational entropy - opposes
protein folding, must be compensated for
by:
• The Hydrophobic Effect - favors protein
folding
• Internal weak interactions:
– Charge-charge interactions (salt bridges)
– Hydrogen bonds
– van der Waals interactions
Distribution of Hydrophobic and
Hydrophilic Residues
Cytochrome c
Red: hydrophobic
Green: hydrophilic
The Hydrophobic Effect in Protein Folding
•Dissolving a hydrophobic group in water solvent forces the water
molecules to form an ordered, low-entropy (“clathrate”) cage around
the hydrophobic group.
•However, entropy of water increases when hydrophobic residues
separate from the water environment and become buried in the interior
of protein.
•Since hydrophobic residues separate spontaneously from water and
form protein’s interior, location of hydrophobic residues in sequence
will help to determine structure of folded protein.
Hydrophobicity of Different Amino Acid
Sides
Hydropathy scales
Hydropathic Index Plot for Bovine
Chymotrypsinogen
Hydrogen Bonding in a Typical Protein
•Interior of a protein is mainly a
hydrophobic environment.
•Partial or full charges of polar
groups in the interior are
neutralized.
•Can involve backbone/
backbone, backbone/side chain
and side chain/side chain
interactions.
•Formation of backbone/
backbone hydrogen bonds to
neutralize backbone partial
charges in the interior of a
protein a major factor driving
formation of secondary structure
in the first place.
Lysosyme
Prediction of Secondary Structure from
Primary Structure (Sequence)
Prediction of Secondary Structure
Adenylate kinase (N-terminal 24 residues)
Bovine pancreatic
trypsin inhibitor
Thermal Denaturation of Ribonuclease A
The Protein Folding Problem: Levinthal’s
Paradox
• Ribonuclease A (124 residues) can potentially form
about 1050 conformations. If it tries a different
conformation every 10-13 seconds, it would take
1050/1013 = 1037 seconds or ~1030 years to try all
conformations, yet ribonuclease A folds in ~1 minute.
• There must be pathways of folding with sequential,
dependent steps (intermediates), instead of a random
“sampling” of all possible conformations.
Energy Surfaces to Visualize Protein
Conformations
Thermal Denaturation of Bovine
Pancreatic Trypsin Inhibitor
Refolding and Disulfide Bond Formation
How to Form the Right Disulfide Bonds:
Protein Disulfide Isomerase
The GroEL-GroES Chaperonin
Other molecular
chaperones:
heat shock proteins
(HSPs)
The Prion Protein: Protein Folding and
Disease
Prion proteins can exist in two
main conformations, one of
which causes prion- based
diseases such as Mad Cow
Disease.
Levels of Protein Structure
• Primary Structure - amino acid sequence in a
polypeptide
• Secondary Structure - local spatial arrangement of a
polypeptide’s backbone atoms (without regard to side
chain conformation)
• Tertiary Structure - three-dimensional structure of
entire polypeptide
• Quaternary Structure - spatial arrangement of
subunits of proteins composed of multiple
polypeptides (protein complexes)
The Four Levels of Protein Structure
Protein Complexes Terminology
• Dimers (2 subunits), trimers (3 subunits), tetramers (4
subunits), etc.
• Polymers - longer, linear or helical multimers
• Homotypic: e.g., homodimer (homotypic dimer) dimer made up of two identical polypeptide chains
• Heterotypic: e.g., heterodimer (heterotypic dimer) dimer made up of two distinct polypeptide chains
Symmetries of Protein Quaternary Structures
Prealbumin Dimer
Two-fold cyclic (C2) symmetry;
isologous interactions
Polymeric Helical Proteins
A Dimer without Symmetry
Heterologous asymmetric binding