Download Lecture 6 Protein Tertiary and Quaternary Structure

BIOC 460, spring 2008
Lecture 6
Protein Tertiary and Quaternary Structure
Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 2, pp. 44-53, 61-62;
Chapter 12, pp. 337-338
Directory of Jmol structures of proteins:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/routines/routines.html
Jmol routine: some structural motifs found in proteins:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/motif/motif.htm
Jmol routine showing locations of hydrophobic and hydrophilic side chains:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/sidechain/sidechain.html
Jmol routine -- 5 different domains in one subunit of pyruvate kinase:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/proteindomains/domain1.htm
Jmol structure of myoglobin:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html
Jmol structures of αβ proteins:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/alpha_beta/alpha_beta.html
Jmol structure of hemoglobin
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/hemoglobin/newhb.html
Key Concepts
• Tertiary and quaternary structures result from folding of primary structure
(and secondary structural elements) in 3 dimensions.
• Tertiary structure
– Most proteins' tertiary structures are combinations of α helices, β
sheets, and loops and turns.
– Larger proteins often have multiple folding domains.
– Folding of H2O-soluble, globular proteins into their native structures
follows some basic rules/principles:
• minimization of solvent-accessible surface area (burying
hydrophobic groups)
• maximization of intraprotein hydrogen bonds
• chirality (right-handed twist and connectivity) of the
polypeptide backbone
• Quaternary structure
– Some proteins have multiple polypeptide chains (quaternary structure).
– Arrangement of polypeptides in multimeric proteins is generally
symmetrical.
– Quaternary structure can play important functional roles for multisubunit proteins, especially in regulation.
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Learning Objectives
• Outline 3 principles guiding folding of water-soluble globular proteins
and the generalizations about protein structure resulting from those
principles. Relate the principles to real protein structures.
• Explain the term amphipathic, with an amphipathic protein α helix as an
example.
• Recognize examples (ribbon diagrams) of such common folding motifs
(frequently encountered combinations of secondary structures) as coiled
coils of α-helices, stacked β-sheets, βαβ elements, β-barrels, and β
saddles.
• Explain the term tertiary structure.
• Define the terms domain and subunit as they relate to protein structure.
Be able to recognize different domains in a ribbon diagram of a single
polypeptide chain with 2 or more domains.
• Describe in general terms the structure of the polypeptide chain of
myoglobin.
Learning Objectives, continued
• Describe the structure of the “immunoglobulin fold” (single domain).
• Describe the general structure of an αβ barrel, including where in the
structure you would expect to find hydrophobic groups and where you
would expect to find polar/charged groups.
• Describe the general structure (arrangement of hydrophobic vs. polar R
groups) of a globular protein that is embedded in a lipid bilayer
(membrane).
– Specifically, describe how the primary and secondary structures of a
bacterial porin relate to the tertiary structure (and function) of a single
porin subunit.
• Explain the term quaternary structure (of a protein), and be able to
describe a protein in terms like "homotetramer", "heterodimer", etc.
• Explain simple rotational symmetry for an oligomeric protein such as a
homodimer like the Cro protein or a heterotetramer like hemoglobin.
– Be able to use (correctly) the terms "2-fold", "3-fold", etc. to refer to
simple rotational axes of symmetry and recognize that simple level of
symmetry in a protein structure.
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Quaternary Structure
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Tertiary Structure
• 3-dimensional conformation of a whole polypeptide chain
in its folded state (includes not only positions of backbone
atoms, but of all the sidechain atoms as well)
• Most water-soluble and membrane proteins are globular
(compact and roughly spherical).
• 3-D structures determined by
– x-ray diffraction of protein crystals, or
– NMR spectroscopy of protein in solution (for proteins that aren’t too
large).
• Every protein has a unique three dimensional structure made
up of a variety of helices, β-sheets and non-regular regions,
which are folded in a specific manner.
3 principles guiding folding of H2O-soluble, globular
proteins & consequences of those principles
• Generalizations about H2O-soluble, globular protein structure
– *minimization of solvent-accessible surface area
– maximization of hydrogen bondin within the protein
– chiral effect
* 1. minimization of solvent-accessible surface area
• burying as many hydrophobic groups as possible
• the most important driving force in folding of water-soluble proteins
• Globular protein structures generally tightly packed, compact units
• Secondary structural elements (α-helices and β sheets) often
amphipathic
– R groups on one side hydrophobic (and face interior of protein)
– R groups on other side hydrophilic (and face aqueous environment,
outside)
• Jmol routine showing locations of hydrophobic and hydrophilic side
chains
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/sidechain/sidechain.html
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1. Minimizing surface (burying hydrophobic side chains)
• Amphipathic secondary structural elements
• Burial of hydrophobic R groups away from H2O requires at least 2
interacting secondary structural elements, e.g., 2 α helices, or a β-α-β
loop (uses α helix to connect 2 parallel β strands), or 2 β sheets, etc.
• How can 2 α helices get together to bury
hydrophobic R groups, if there's water
around them?
amphipathic helices -- used to
bury hydrophobic R groups toward
interior of protein on 1 side of helix
while other side of helix interacts
with H2O
Berg et al., Fig. 2.44
α-helical coiled coil (2 α helices coiled around each
other) of a leucine zipper motif (heptad repeat)
• schematic diagram ("helical wheels" projections down helix axes)
Fig. 16-30 from Stryer,
Biochemistry, 4th ed.(1995)
• Residues a and d of each strand pack tightly together to form a
hydrophobic core.
• If residues b, c, and f on periphery are polar or charged, the helices are
amphipathic helices.
• Note: Any protein α-helix will be amphipathic if one side of the helix is in
a polar environment and the other side is in a hydrophobic environment.
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2. Maximizing hydrogen bonds within the protein
• especially important in "driving"/stabilizing formation of
secondary structures like α-helices and β sheets
– makes “burying” polar N-H and C=O groups of
backbone in interior of protein more favorable
thermodynamically
• polar side chains sometimes also buried, if their polar
groups are hydrogen-bonded
• Polar backbone groups and side chains tend to be either
– in contact with water (hydration)
OR
– hydrogen-bonded with OTHER PROTEIN GROUPS (e.g.,
in secondary structures like α-helices and β sheets)
3. the chiral effect
• tendency of extended backbone structural arrangements to be righthanded as a result of having all L-amino acids
• Consequences: twist and connectivity
– twist:
• α helices of L-amino acids tend to be right-handed.
• β-conformation strands (and sheets) of L-amino acids tend to
twist in a right-handed direction, forming saddles or barrels.
– connectivity:
• crossovers between adjacent secondary structural elements, e.g., in
βαβ structure, are usually right-handed.
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Structural motifs
• recognizable patterns of combinations/groupings of secondary
structural elements
• bury hydrophobic R groups in between “layers”/elements
• Examples of motifs:
– coiled coils of 2 or more α helices (αα)
– stacks of β-sheets
– βαβ elements
– β barrels (β sheet folds/twists into a cylinder)
– β saddles (twisted β sheet)
• Jmol routine: some structural motifs found in proteins
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/motif/motif.htm
• Some motifs have functional significance, such as the helix-loop-helix
(helix-turn-helix) DNA-binding motif or the EF hand calcium-binding
motif. Others serve only a structural role.
water-soluble globular protein tertiary structures
Examples:
1. Myoglobin (Mb):
• Jmol structure of Mb:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html
•
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•
•
•
•
•
•
binds O2 in muscle cells for storage and for intracellular
transport, using a heme group
mostly (70%) α-helical; rest mostly turns & loops (at
surface)
first high-resolution crystal structure of a protein ever
determined
very compact structure (almost no empty space inside)
very water-soluble
5 Pro residues, 4 in turns
8 α helices, designated by letters A - H, from N to C
terminus
Helices amphipathic (surface sides hydrophilic R groups,
buried sides more hydrophobic R groups)
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Myoglobin
structure
Berg et al., Fig. 2.48B;
heme black with
purple Fe2+
Nelson & Cox, Lehninger Principles of
Biochemistry, Fig. 4-16
(heme in red; blue residues: Leu, Ile, Val, Phe)
Myoglobin structure, continued
• Distribution of Amino Acids in Mb structure
• (hydrophobic residues in yellow, charged residues in blue, others in
white)
A. surface view ; B. cross-sectional view showing interior of protein
• NOTE: many charged residues on surface, none in interior
• many hydrophobic residues in interior, but also a few on surface
• The only polar residues inside are 2 His residues involved in binding the
heme and O2.
Berg et al., Fig. 2-49
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2. Triose phosphate isomerase, an αβ barrel protein
• an enzyme in the glycolytic pathway)
• Jmol structures of αβ proteins:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/alpha_beta/alpha_beta.html
• an (αβ)8 barrel (parallel 8-stranded β barrel on interior, surrounded by
α helices, a structural motif found in many different enzymes
• Some βαβ enzymes (triose phosphate isomerase, and one domain of
pyruvate kinase):
•Garrett & Grisham, Biochemistry, 3rd ed., Fig. 6-30
3. Protein Domains
• Domains: structurally independent folding units looking like separate
globular proteins but all part of same polypeptide chain
• connected in same primary structure
• Larger proteins often have 2 or more domains.
• Jmol routine -- 4 different domains in one subunit of pyruvate kinase:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/proteindomains/domain1.htm
•Troponin C, a protein
found in muscle
•2 domains, all one polypeptide chain
Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 4-19
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3. Protein domains, continued
• the “immunoglobulin fold”, a “β sandwich” domain
• a cell surface protein (CD4), with 4 similar domains (each in a
different color).
– The folding motif of each of the 4 domains is the same.
• Each domain consists of 2 antiparallel β sheets, with loops between β
strands: motif = the "immunoglobulin fold".
Berg et al., Fig. 2-52
4. Porins (example of a membrane protein)
• found in outer membranes of bacteria and
in outer mitochondrial membranes
• channel-forming proteins - permit passage
of ions and small molecules across
membrane
• globular, but their "solvent" is NOT water it’s a membrane
• lipid core of membrane like a very
nonpolar solvent
• structure of each chain of porin mainly a
large β barrel (big antiparallel β sheet,
16 strands, folded into a cylinder)
• structure sort of like an "inside out" watersoluble protein
• hydrophobic residues on outer surface,
interacting with hydrophobic lipid core of
membrane
• inner side of the barrel forms water-filled
channel across membrane; has more
hydrophilic (charged and polar) R groups
Berg et al., Fig. 2-50
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Structure of one subunit of a bacterial porin
• left: side view, in plane of membrane; right: view from periplasmic space
(from inside, looking out through pore in outer membrane)
Berg et al., Fig. 12-20
On a single strand in β conformation,
where are the side chains of the amino acid
residues? (all on one side? Alternating sides?
3 residues’ R groups on one side, 1 on the other side?)
Amino acid sequence of a porin
• β strands are indicated, with diagonal lines indicating direction of
hydrogen bonding along the β sheet
• hydrophobic residues (F, I, L, M, V, W and Y) shown in yellow
Berg et al., Fig. 12-21
•Note the more or less alternating hydrophobic and hydrophilic residues
in the β strands (adjacent R groups project out from sheet on opposite sides).
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Quaternary structure (4° structure)
• 3-dimensional relationship of the different polypeptide chains
(subunits) in a multimeric protein, the way the subunits fit together
and their symmetry relationships
• only in proteins with more than one polypeptide chain; proteins with
only one chain have no quaternary structure.)
Terminology
• Each polypeptide chain in a multichain protein = a subunit
• 2-subunit protein = a dimer, 3 subunits = trimeric protein, 4 = tetrameric
• homo(dimer or trimer etc.): identical subunits
• hetero(dimer or trimer etc.): more than one kind of subunit (chains with
different amino acid sequences)
• different subunits designated with Greek letters
– e.g., subunits of a heterodimeric protein = the "α subunit" and the "β
subunit".
– NOTE: This use of the Greek letters to differentiate different
polypeptide chains in a multimeric protein has nothing to do
with the names for the secondary structures α helix and β
conformation.
• Some protein structures have very complex quaternary arrangements;
e.g., mitochondrial ATP synthase, viral capsids….
Examples of quaternary structure in proteins
• Cro protein from
bacteriophage lambda (λ), a
homodimer
Berg et al., Fig. 2-53
• Hemoglobin, a heterotetramer
(α2β2)
• 2 identical α subunits (red)
structurally similar to 2 identical
β subunits (yellow)
• α and β also very similar to
structure of myoglobin (both
primary and tertiary structure)
• gene duplication of single
ancestral gene and subsequent
divergent evolution of
sequences --> different globin
genes
• tertiary "fold" conserved through
evolution
Berg et al., Fig. 2-54
• Jmol structure of hemoglobin
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Symmetry in quaternary structures
• simplest kind of symmetry = rotational symmetry
• Individual subunits can be superimposed on other identical subunits
(brought into coincidence) by rotation about one or more rotational
axes.
• If the required rotation = 180° (360°/2), protein has a 2-fold axis of
symmetry (e.g., Cro repressor protein above).
• If the rotation = 120° (360°/3), e.g., for a homotrimer, the protein has a
3-fold symmetry axis.
Rotational symmetry in proteins:
Cyclic symmetry: all subunits are
related by rotation about a single
n-fold rotation axis (C2 symmetry
has a 2-fold axis, 2 identical
subunits; C3 symmetry has a
3-fold axis, 3 identical subunits, etc.)
What type of rotational axis of
symmetry is apparent in the
hemoglobin structure above?
LEC 6, Protein Tertiary and
Quaternary Structure
Nelson & Cox, Lehninger Principles of
Biochemistry, 4th ed., Fig. 4-24a
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