Download 3-D Structure of proteins

3-D Structure of proteins
Outline
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There are 4 levels of protein structure
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Methods for determining protein structure
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Secondary structure of proteins
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The a Helix
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b Structure
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Other conformations are found in globular proteins
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Tertiary structure of globular proteins
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Quaternary structure
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Protein folding and stability
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Protein folding is assisted by Chaperones
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Protein denaturation and renaturation
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Collagen, a Fibrous Protein
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Myoglobin, a Globular Protein
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Hemoglobin, a globular protein possessing quaternary structure
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Oxygen binding to myoglobin and hemoglobin
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Oxygen-binding curves of myoglobin and hemoglobin
• Proteins are the main players in the life of a cell. Each protein is a unique
sequence of amino acid residues, each of which folds into a unique, stable,
three dimentional structure that is biologically functional. Proteins are the
most structurally and functionally diverse group of macromolecules. Their
vaired functions stem from some key features:
• 1. Proteins are linear polymers built of building blocks clalled amino acids.
– unlimited order of amino acids makes unlimited variety of proteins.
– the fundamental amino acid alphabet in proteins is several billion years
old.
– each protein is a unique sequence of amino acids residues linked by
peptide bonds.
• peptide bonds are very stable (in absence of an enzyme, peptide
bond can last 1000's yrs)
• 2. Proteins contain a wide range of functional groups.
– hydroxides, thiols, thioesaters, carboxylic acids, amino groups,
phosphates, etc...
– reactivity of these groups accounts for enzyme activity.
• 3. Proteins can interact with one another and with other biological
molecules to form complex assemblies. Molecular cooperation leads to
molecular synergism ( proteins in a group act synergistically to generate
capabilities not afforded by the individual component proteins).
• Conformation = spatial arrangement of atoms
that depends on rotation of bonds. Can change
without breaking covalent bonds.
• Since each residue has a number of possible
conformations, and there are many residues in a
protein, the number of possible conformations
for a protein is enormous.
• Native conformation = single, stable shape a
protein assumes under physiological conditions.
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In native conformation, rotation around covalent bonds in polypeptide is
constrained by a number of factors ( H-bonding, weak interactions, steric
interference)
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Biological function of proteins depends completely on its conformation. In biology,
shape is everything.
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Proteins can be classified as globular or fibrous.
Some proteins are quite rigid, while other show limited flexibility
rigid proteins are often structutral proteins
flexibility is useful in making hinges, springs, and levers
There are 4 levels of protein structure
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Primary structure
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linear sequence of amino acids
determined by nucleotide sequence of gene
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held by covalent forces
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primary structure determines overall shape of folded polypeptides (i.e primary structure determines
secondary , tertiary, and quaternary structures).
Secondary structure
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regions of regularly repeating conformations of the peptide chain (a helices, b sheets)
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maintained by H-bonds between amide hydrogens and carbonyl oxygens of peptide backbone.
Tertiary structure
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completely folded and compacted polypeptide chain.
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stabilized by interactions of sidechains of non-neighboring amino acid residues (fibrous proteins lack
tertiary structure)
Quaternary structure
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association of two or more polypeptide chains into a multisubunit protein.
Levels of Protein Structure
Interactions in Folding
Protein with itself :
Hydrogen bonds
Electrostatic interactions
Van der Waals interactions
Protein-Solvent: induces burial of hydrophobic residues
Hydrophobic core
Hydrophobic residue
Hydrophilic residue
Secondary structure of proteins
Secondary structure is the hydrogen-bonded arrangement of the
backbone of the protein. The two most common secondary
structures are the a-helix and b-strands (many strands make sheets)
• The a Helix
most frequently observed secondary structure.
• virtually always found as right-handed
• generally have 3.5-3.7 residues per turn:
• pitch (advance per turn) = 0.54 nm
• rise (advance per amino acid residue) = 0.15 nm
• each carbonyl oxygen (residue n) of polypeptide backbone H-bonded to
backbone amide hydrogen of the fourth residue toward C- terminus (n +
4). Therefore first 3 and last 3 residues lack H-bonding partners within
helix)
• each hydrogen bond closes a loop of 13 atoms
• hydrogen-bonds, which stabilize helix, are nearly parallel to long axis of
helix; all carbonyl groups point toward C-terminus.
• cumulative effect of many H-bonds within a helix stabilizes this
conformation, especially in hydrophobic regions in the interior of the
protein, where there are no water molecules to compete for H-bonds.
• side chains of amino acids in a helix point outward from the cylinder of the
helix.
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Side chains affect stability of helix:
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alanine has small , uncharged side chain, so fits well within helix, and is often found in helices of
globular proteins.
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glycine, has -H side chain, and destabilizes helices because rotation around a-carbon is so
unconstrained
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proline never found in interior of a-helices. It has rigid cyclic side chain which causes steric
interference. Also, lacks amide hydrogen for H-bonding with carbonyl oxygen of other a.a. in
helix.
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a-helices usually end with structures known as helix stop signal (e.g. capping box).
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side chain of N-terminal residue (n) of helix interacts with backbone amide hydrogen of fourth
residue of helix, resulting in a conformation whose f and y angles are not compatible with
formation of a helix.
– Serine and threonine usually found at initial positioning of capping box, and glutamate is
often the fourth residue.
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Globular proteins vary in their a-helical content:
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in myoglobin, 75% of residues found in a-helices
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chymotrypsin has very little
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average for globular proteins is 26%.
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Amphipathic helices = have hydrophilic amino acids on one face of helix cylinder and
hydrophobic amino acids on opposite face. Such amphipathic helices often located on surface of
globular proteins.
β Structure
• portions of the polypeptide chain that are almost fully extended
• includes b strands and b sheets. b sheets consist of multiple b strands
arranged in sheets.
• stabilized by H-bonding between carbonyl oxygens and amide hydrogens
on adjacent b strands (of same or different polypeptide)
• β strands can be parallel or antiparallel. Parallel strands less stable
because of distorded H-bonds.
• in β sheets, side chains point alternately above and below plane of
sheet.
• β sheets may contain 2-15 strands, and can form barrel shaped
structures
• many globular proteins contain regions of b structure. Rubredoxin
composed of 7 b strands arranged in 3 antiparallel b sheets.
• as in a helices, side of b sheets facing interior is hydrophobic, and surface
side is hydrophilic. In cases where entire sheet is in interior, all amino
acids composing sheet are hydrophobic.
Other conformations are found in globular proteins
• Nonrepetitive regions
– sometimes called random coils. but are actually stable, highly
ordered structures.
– connect secondary structures; provide directional changes necessary
for proper folding (Loops, turns, and hairpin loops) (Fig 4.7).
– most globular proteins have many residues in nonrepetative
conformations (as much as in helices and strands)
– least conserved areas of protein
– hairpin loops in the variable domains of the antibody molecule bind to
antigens. High mutation rates observed function to generate antibody
diversity. This process, in conjunction with alternative splicing of
genes, allows an organism with a finite number of antibody genes to
generate an almost unlimited number of antibodies capable of
recognizing. a very diverse population of antigens.
• Motifs
• supersecondary structures; recognizable
combinations of a helices and b strands
that appear in a number of different
proteins (Fig 4-8 and 4.9).
• sometimes have a particular function
a. helix-loop-helix
b. bab unit
c. hairpin
d. Greek key
Globular proteins:
• water soluble; compact, spherical; hydrophobic interior and hydrophilic
surface.
• indentations specifically and transiently bind other compounds (active and
allosteric sites)
• most enzymes are globular
• Most proteins are globular. Its the focus of this chapter.
Fibrous proteins:
• static molecules; provide mechanical support
• typically water insoluble
• usually built on single repetitive structure assembled into cables or
threads e.g. a-keratin, collagen, silk.
Tertiary structure of globular proteins
• result from folding of polypeptide into closely packed, nearly spherical
shape.
• stabilized primarily by noncovalent interactions (mostly hydrophobic
effect) between side chains of amino acid residues.
• Lots of variety in tertiary structure.
• many globular proteins composed of independently folded globular units
called domains. Domains consist of combinations of motifs (range in size
from 30-300 a.a.)
a. β meander
b. α/β barrel
• small proteins usually contain one domain. Larger proteins may contain
more than one.
• often, each domain has a particular function. In multifunctional enzymes,
each catalytic activity associated with different domain.
• regions between domains often form crevices that serve as binding sites
(e.g. antibody proteins).
The forces that give rise to the tertiary structure of a
protein are
• Ionic bonding
• hydrogen bonding
• hydrophobic interaction
• disulfide bonds
Quaternary structure
Limited to proteins with multiple subunits.
• Each subunit called a monomer. A multisubunit protein is called an
oligomer.
• subunits in an oligomeric protein always have defined stoichiometry.
• Monomers may be identical or different.
• when several metabolic reactions are catalyzed by oligomer, its called a
multienzyme complex.
• Subunits of oligomer usually held together by noncovalent forces.
Usually hydrophobic interactions, but electrostatic forces may also be
involved.
A large proportion of globular proteins have quaternary structure.
Probable reasons are:
1. more stable than dissociated subunits
2. active sites of some oligomers formed by amino acids on adjacent
polypeptides.
3. likelyhood of errors greater for long polypeptides. Therefore more
efficient to synthesize several smaller subunits
4. 3D-structure changes when proteins bind ligands. Critical step in
biological activity of many proteins (i.e regulatory function)
Denaturation
The natural or native structures of proteins may be
altered, and their biological activity changed or
destroyed by treatment that does not disrupt the
primary structure. This denaturation is often done
deliberately in the course of separating and
purifying proteins. For example, many soluble
globular proteins precipitate if the pH of the solution
is set at the pI of the protein. Also, addition of
trichloroacetic acid or the bis-amide urea
(NH2CONH2) is commonly used to effect protein
precipitation. Following denaturation, some proteins
will return to their native structures under proper
conditions; but extreme conditions, such as strong
heating, usually cause irreversible change.
Some treatments known to denature proteins are
listed in the following table.
Denaturing Action
Mechanism of Operation
• Heat hydrogen bonds are broken by increased
translational and vibrational energy.
(coagulation of egg white albumin on frying.)
• Ultraviolet Radiation Similar to heat (sunburn)
• Strong Acids or Bases salt formation; disruption of
hydrogen bonds.
(skin blisters and burns, protein precipitation.)
• Urea Solution competition for hydrogen bonds.
(precipitation of soluble proteins.)
• Some Organic Solvents
(e.g. ethanol & acetone) change in dielectric constant
and hydration of ionic groups.
(disinfectant action and precipitation of protein.)
• Agitation shearing of hydrogen bonds.
(beating egg white albumin into a meringue.)