Protein Structurefunction: Lecture contents

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Protein Structure-function Lecture contents
IV. Amino Acids and their Chemical
Properties V Peptides Levels of Protein
Structure -Secondary Structure ?-helices,
?-sheets and ?-turns -Tertiary and Quaternary
Structure Coiled-Coils etc VI. Protein
folding Examples of protein Structure and
function VII. Enzymes VIII. Transport
proteins (hemoglobin)
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Lecture IV Mathews ( p126-147, p161-193)
IV. Amino Acids and their Chemical
Properties Classification Special
amino-acids G,P,C. Structures Important you
will have to know them for the QUIZ Properties
Weak acids bases, Zwitterion Peptide bond
Define backbone of polypeptides Primary
Structure of polypeptide. An example insulin
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Figure 5.1 3D Structure of Myoglobin
a-helix
- first to be determined by x-ray crystallography
Heme ligand
a-amino-acids Side chain
- revealed how the protein bound heme (loaded
with oxygen) and gave the first detailed look at
a protein structure
- now over 2000 protein structures are known
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Protein Structure-function Lecture contents
V Peptides Levels of Protein Structure -Second
ary Structure ?-helices, ?-sheets and
?-turns -Tertiary and Quaternary Structure
Coiled-Coils etc VI. Protein folding Examples of
protein Structure and function VII.
Enzymes VIII. Transport proteins (hemoglobin)
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Figure 6.27 - Levels of Protein Structure
Peptide bond Backbone interactions Side chains
interactions Side chains interactions Electrostat
icvan der waals
The folded protein structure is stabilized by a
variety of weak chemical interaction, and in
some cases covalent (disulfide) bonds between
cysteine residues
Disulfide bond
R CH2SSCH2R
Cys Cys
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Protein structure overview
Structural element Description primary
structure amino acid sequence of protein
secondary structure helices, sheets,
turns/loops super-secondary structure associatio
n of secondary structures domain self-containe
d structural unit tertiary structure folded
structure of whole protein includes
disulfide bonds quaternary structure assembled
complex (oligomer) homo-oligomeric (1
protein type) hetero-oligomeric (gt1
type)
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Peptide bond formation
Glycine(G)
Alanine(A)
The peptide bond has partial double bond character
-
O
C ter

N ter
C
N
H
Resonant Structures
Glycylalanine (GA)
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Planar Character of a Peptide Bond
Figure 5.12 - partial double bond character
prevents the peptide bond from rotating
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Figure 6.2 rotation about the alpha carbon
- with the lack of rotation around the peptide
bond the point of flexibility along the backbone
of the protein arises at the ? - carbon.
?-carbon
Bonds allowing for rotation along the protein
backbone
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Alpha-Helix b-sheets
Main chain Ca
F, y angle value determine secondary structure
Ribbon
Amide plane
F-57 y-47
a-carbon i4
Hydrogen bond i,i4
Secondary structure involves hydrogen bonding
between atoms of the backbone
a-carbon i
Side chains R outside the Helix
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Protein secondary structure helices
- rod like right-handed
INTRA-chain H-bonds between gtCO group of each
peptide residue and the gtN-H group of the 4th
amino acid away
- alpha helices are about 10 residues on average
H-bonding in a-helix
- side chains of alpha-helices are well
staggered, preventing steric hindrance
- helices can form bundles, coiled coils, etc.
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Helical Wheels
- a tool to visualize the position of amino acids
around an alpha-helix - allows for quick
visualization of whether a side of a helix posses
specific chemical properties - example shown is
a helix that forms a Leucine-Zipper
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?-Helix Breakers
Most amino acids like to be in an
?-helix. Notable exceptions GLYCINE PROLINE
(Imino Acid)
No Hydrogen On this N to H-Bond
O
C-O
N
H
- proline residues often serve as ?-Helix
Breakers - often found at the boundaries of
?-Helices and in turns
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Proteins with ?-helices
Major structural component in many proteins, some
globular proteins contain mostly ?-helices,
connected by turns (i.e., hemoglobin 70
?-helices)
Some Interesting ?-Helices - small DNA binding
helices - membrane spanning helices -
amphipathic helices - coiled Coils
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DNA Binding - an ??-helix fits perfectly into the
major groove of double stranded DNA. - many DNA
binding proteins use particular ??-helices to
specifically recognize a DNA sequence.
dsDNA
Membrane Spanning - contains hydrophobic amino
acids in the central region to allow the protein
to cross a bi-layer membrane
Hydrophilic Hydrophobic
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Amphipathic Helices Amphipathic hydrophilic
hydrophobic - these helices posses hydrophilic
amino acids on one side and hydrophobic residues
on the other. - these ?-helices in some cases
can be used to associate a protein to a membrane.
Hydrophobic
Hydrophilic
hydrophilic head group
aliphatic carbon chain
lipid bilayer
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6.13 Coiled Coils a higher order structure
composed of alpha-helices (supersecondary
structure) ex Collagen.
Alpha-Helix
A Triple Coiled Coil
Axis for Interaction with other alpha-helix. In a
double coiled-coil.
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Nter
Cter
F-57 y-47
F-139 y135
a Carbon
Carbon (C0)
a Carbon
Cter
Nter
Alpha-Helix Beta-Sheet (antiparallel)
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Protein structure beta-sheets
- the basic unit of a beta-sheet is called
a beta-strand
N
O
C
- repeating unit like the alpha helix
- beta-sheets can form various higher-level
structures, supersecondary structure such as a
beta-barrel
parallel
twisted
anti-parallel
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The Beta-Sheets

• - strands of amino acids held together in sheets
  by INTER-STRAND H-Bonding
• - bonding between backbone gtCO and gtN-H on
  different strands
• strands of the b-sheets tends to be twisted and
  inclinated in a b-barrel
• - the R-groups lie perpendicular to the sheets
  stick out on either face of the sheet

In a b-barrel the amino acids side chains inside
the barrel are very often b-branched or
hydrophobics
R
R
R
R
R
R
R
R
R
R
R
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Beta-Sheets and DNA
- an alpha-helix is of appropriate size to fit in
the major groove of DNA - beta sheets fit very
well into the minor groove of DNA double
helices - beta-sheets can also used in DNA
binding but are generally less commonly used
Alpha-Helix
Beta-Sheet
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Figure 6.12 An example of complex
beta-sheets Silk Fibroin - multiple pleated
sheets provide toughness rigidity to many
structural proteins.
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Protein structure turns/loops
beta-sheet
alpha-helix
- there are various types of turns, differing in
the number of residues and H-bonding pattern
- loops are typically longer they are often
called coils and do not have a regular, or
repeating, structure
loop (usually exposed on the surface of proteins)
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Beta - Turns
Figure 6.18
There are two classes of beta-turns - type I -
type II Note the position of R2 and R3 in both
cases Type I turns have the amino acids side
chains on the same side. Type II turns have the
amino acids side chains on the opposite sides.
Note H-bonding between backbones of residue 1
4
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Gamma-Turns
Proline
A 3 amino acid turn utilizing proline at the
turn. H-bonding with CO of residue 1 and N-H of
residue 2
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Levinthal paradox
in vitro
in vivo
denatured protein random coil 106
possible conformations
folding
folding
Native protein 1 stable conformation
t seconds
t seconds or much less
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Levinthal paradox a new folding view is needed
Consider a protein with 100 amino acids - using a
very simplified model where there are only 3
possible orientations per residue
- assume 1 conformation can form every 10-13
seconds (100 picoseconds) - then 5 x 1047
x 10-13 s 1.6 x 1027 years to correctly fold a
protein Obviously NOT ALL conformations can be
sampled during folding!
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Resolving it
a. there are a limited number of secondary
structural elements b. these elements tend to
form spontaneously during the co-translational
folding of a protein c. proteins fold via
so-called folding landscapes, where the
proteins follow pathways of folding that lead
to the correct three-dimensional structure d.
folding intermediates may be important in such
folding landscapes/pathways
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Protein folding theory
limited number of secondary structure elements
helices, sheets and turns
folding can be thought to occur along energy
surfaces or landscapes
Dobson, CM (2001) Phil Trans R Soc Lond 356,
133-145
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A simplified view A folding funnel
unfolded (non-native) states (two of many
different conformations are shown)
Energy landscape - descent towards Free energy
minimum state - A- rapid folding B-
secondary energy minima
Native state (N)
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Protein folding Thermodynamic
The overall free energy change on folding is
negative so favorable. Free energy is a balance
of several thermodynamic factors -Conformational
entropy Unfavorable energy favor random chains
conformation due to the burying of hydrophobic
residues interacting with water. -Enthalpy
contribution Favorable energy from
intramolecular side groups interaction. -Entropy
contribution from hydrophobic effect Favorable
energy due to the burying of hydrophobic R group
Energy balance
? G ? H - T ? S
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Proteins fold in stages
Local folding through nucleation of small
clusters of residues due to the hydrophobic
effect A General Order of Folding (Mathews
p181-190). 1. Random polypeptide hydrophobic
residues stabilized by water forming a cagelike
structure, DS low. 2. Secondary structure start
to form -hydrophobic effect (hydrophobic
residues buried inside the protein
-gtfavorable energy DS). -release of water
due to hydrophobic effect-gt randomness
conformational entropy increase (unfavorable
energy DS). 3. Secondary structure are formed,
domains, protein folded Intramolecular side
chain interactions create an enthalpy negative
favorable energy
folded protein
polypeptide
secondary structures
domains
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Molecular chaperones
- the great majority of proteins can fold without
assistance, in a co-translational manner - some
proteins, which may have difficulties reaching
their native states, must be stabilized by
molecular chaperones by assisted folding - bind
to nascent (emerging) polypeptides and stabilize
them mostly by binding hydrophobic residues -
otherwise these hydrophobic residues tend to
associate with other hydrophobic residues,
leading to intra- or inter-molecular associations
with other proteins that prevent proper
folding - there are dozens of different types of
molecular chaperones, and some accomplish
functions different from helping protein
folding - e.g., some help protein assembly, some
help to transport proteins to various parts of
the cell, some help damaged proteins from
refolding
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Co-translational protein folding
definition a process which occurs during the
translation (synthesis) of a protein on the
ribosome
- the first 30 amino acids of the polypeptide
chain present within the ribosome is
constrained (the amino, or N-terminus emerges
first, and the C-terminus emerges last) - as
soon as the nascent chain is extruded, it will
start to fold co-translationally (i.e., acquire
secondary structures, super-secondary structures,
domains) until the complete polypeptide is
produced and extruded
NH3
folding
assembly
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GroEL-GroES
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Figure 6.16 Examples of Some Globular Proteins
- viewing proteins as secondary structure models
parallel beta-barrel - alpha/beta sequence
beta-sandwich
helix bundles
twisted beta-sheet
anti-parallel beta-sheet
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Protein domains
- many proteins have several compact globular
regions. Each globular unit is called a
domain. - domains tend to have 50 up to 200-300
amino acids - less than 50 is difficult to fold
stably - more than 300 is difficult to fold
correctly - a single domain is typically made of
a single stretch of primary sequence there are
many exceptions though
Hydrophilic
domain 1
domain 2
Hydrophobic Core
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Protein tertiary structure
- folding and packing of secondary structure
elements, super-secondary structure elements,
domains - tertiary folding is stabilized by the
same non-covalent interactions as found in the
secondary structures but involve amino-acid side
chain interactions only not main chain atoms
interactions. - H-bonding - ionic interactions -
van der Waals forces - hydrophobic
interactions - can be stabilized by covalent
bonds di-sulphide linkages (Bonds)
-Cys
-S
-Cys
-Cys
-S
Oxidizing agent
-Cys
CystEine
Cystine
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Protein quaternary structure
Association of multiple polypeptides into a
functional unit - many, although not all
proteins engage in this - individual proteins in
the quaternary structure are calledsubunits Ex
ample prefoldin (a so-called molecular
chaperone that assists the co-translational
stabilization of proteins during their folding
in vivo (in the cell)
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Prefoldin quaternary structure
two types of proteins (subunits) assemble into
a hexamer (6 subunits)
- structure of prefoldin hexamer -
oligomerization (assembly) domain is a double
beta-barrel structure composed of beta-strands -
coiled coils consist of two helices winding
around each other
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Symmetries of ProteinQuaternary Structure
Figure 6.30