Biochemistry-2_2-proteins 1
Biochemistry
2. 2 Protein
structure and
function
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Protein structure & function
Proteins are the most versatile
macromolecules in living systems,
and serve crucial functions in essentially
all biological processes
3.1 Proteins are built from a repertoire of 20 amino acids
3.2 Primary structure: amino acids linked by peptide bonds form
polypeptide chains
3.3 Secondary structure: polypeptide chains fold into regular
structures such as alpha helix, beta sheet, & turns & loops
3.4 Tertiary structure: water-soluble proteins fold into compact
structures with nonpolar cores
3.5 Quaternary structure: polypeptide chains can assemble into
multisubunit structures
3.6 The amino acid sequence of a protein determines its
three-dimensional structure
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Proteins - Key properties - a wide range of
functions
1. Proteins are linear polymers built of monomer units called
amino acids - spontaneously fold into 3-dimensional structures
2. Proteins contain a wide range of functional groups - alcohols,
thiols, thioethers, carboxylic acids, carboxamides, & a variety
of basic groups - eg. chemical reactivity essential to function
of enzymes
3. Proteins can interact with one another, & with other biological
macromolecules to form complex assemblies macromolecular
machines
4. Some proteins are quite rigid, whereas others display limited
flexibility - structural elements in the cytoskeleton v parts that
act as hinges, springs, & levers etc
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Protein functions (test)
 1) Catalysis – Enzymes (the proteins that direct and accelerate
1000 biochem. reactions, mild condition, temperature)
 2) Structure (structural materials, provide protection and
support, specific properties – collagen, elastin, fibroin)
 3) Movement (all type of cell movement, actin, tubulin, other
cytoskeleton proteins)
 4) Defense (protective role, keratin-skin cells, bloodclotting
proteins-fibrinogen, thrombin; immunoglobullins)
 5) Regulation (peptide hormones insilin, glucagon, growth
hormone)
 6) Transport (carriers of molecules or ion across membrane,
Na+K+ATPase, glucose transporter, hemoglobin, lipoproteins)
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Primary structure charge and the amide nitrogen a partial positive
charge, setting up a small electric dipole.
Virtually all peptide bonds in proteins occur in
this trans configuration; an exception is noted in
Figure 4–8b.
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Trans Cis
Peptide bonds- trans configuration
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Secondary structures of proteins
 Refers local conformation of some part of
polypeptide
Types:
 a helix
 b sheet
 b loop
 Hydrogen bond between carbonyl group
and N-H in polypeptide
Pauling and Corey
predicted the existence of these secondary
structures
in 1951, several years before the first complete
protein
structure was elucidated.
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α - helix
-Rigid rodlike structure,
- right handed coiled springlike
conformation
- 3.6 AA per turn of the helix
-Hydrogen bonds between N-H group
of each AA and CO group of AA four
residues away
- Collagen –prolin – left handed helix
-Charged AA and Try incompatible
with a-helix
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Super helix: alpha helical coiled coil
Can be as long as 1000 Å, very stable
Helical cables in these proteins serve a mechanical role,
forming stiff bundles of fibers
Found in: • myosin and tropomyosin in muscle,
• fibrin in blood clots,
• keratin in hair, quills, claws, hoofs, & horns
• intermediate filaments
(cytoskeleton or internal scaffolding of cells)
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β - sheet
Antiparallel
parallel
-Two and more polypeptide chains
line up side by side,
-Each polypeptide is fully extended
-Hydrogen bonds polypeptide
backbone N-H and CO adjacent
chains
-Parallel (same directions)
-Antiparallel (opposite direction)
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Antiparallel beta sheet
Strands linked by H-bonding between opposite amino acids
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Parallel beta sheet
Strands linked by H-bonding of an aa on one strand to two
different aa on the adjacent strand
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Structure of mixed beta sheet
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A twisted beta sheet, schematic model
Rotated 90 degrees
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Fatty acid-binding protein
Rich in beta sheets
Arrow pointing
to carboxylterminal
end
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Relative frequency of aa in secondary structures
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Alternative conformations: context
alpha helix beta strandSame aa sequence
Tertiary interactions (between residues far apart) affect secondary structures
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Five themes of 3D structure of proteins
1. 3D is determined by AA sequences
2. Function of proteins depends on its structure
3. Isolated protein 1 or small number of stable
struc.forms
4. The most important fources- noncovalent
interaction
5. Common structural patterns
Every protein has a three-dimensional structure
that reflects its function.
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Protein conformation
 Spatial arrangement of atoms in protein
 Rotation about single bound
 Thermodynamically the most stable
 Lower Gibson free energy
 Native proteins
• Proteins are stabilized by multiple WEAK interactions
• Hydrophobic interactions are the major contributors –
globular forms of most soluble proteins
•Hydrogen bonds and ionic interactions are optimized in the
specific structures - Thermodynamically the most stable
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Protein conformation
 Spatial arrangement of atoms in protein, Rotation
about single bound, Thermodynamically the most
stable, Lower Gibson free energy
 Native proteins
• Proteins are stabilized by multiple WEAK interactions
• Hydrophobic interactions are the major contributors – globular forms
of most soluble proteins
• Hydrogen bonds and ionic interactions are optimized in the specific
structures - Thermodynamically the most stable
the three-dimensional structure
 Proteins conformation (covalent bond, free rotation, unlimitied
number of conformations)
 each protein has a specific chemical or structural function,
strongly suggesting that each has a unique three-dimensional
structure
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Proteins tertiary and quartery
structures
 Complete 3D structure of polypeptides
 2 general classes: fibrous and globular
 Fibrous proteins – structural roles, simple repeating elements of
secondary structures
 Globular proteins – complicated, several types of 2nd structure,
myoglobin (1st proteins X-ray)
 Domains- region of proteins which can fold stably and
independently
 Quartery structures: interaction between subunit, promoters
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Interaction –stabilized tertiary structure
 Hydrophobic interaction (hydrophobic R
groups –close proximity, exclusion water,
folding of globular proteins)
 Electrostatic interactions- between ionic
groups – salt bridge
 Hydrogen bond
 Covalent bond (disulfide bridges)
TEST
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Fibrous and globular proteins
 FIBROUS – High proportions of regular secondary
structures (a-helix, b-sheeds)
- long rod-shaped, sheetlike molecules,
- insoluble in water, physically tough, keratins (skin, hair, nails)
 a-keratin, colagen, silk fibroin
- STRUCTURAL, PROTECTIVE FUNCTION
 GLOBULAR
- compact spherical molecules, usually water soluble,
- DYNAMIC function : ENZYMES, immunoglobulin's,
TRANSPORT
- cavities, clefts-complementary to LIGAND
- Hemoglobin-----
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a-keratin
 Hair, wool, skin, fingernails
 A-helical polypeptides
 3 a helical chains –left handed supercoiled structure –
protofibril
 Microfibril
 Macrifibril
 AA- no prolin, ala, leu; R outside- wather insoluble; hard
keratins- disulfide (oxidizing)…
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 trvalá ondulace
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Collagen
 Abundant protein in vertebrates
 Connective tissue cells, secreted to extracellular
matrices
 In structures: skin, bones, tendors, blood vessels
 3 left handed polypep. helices, twisted around each
other – righ handed superhelix
 AA – 30% glycine, 30% prolin, 4-hydroxyprolin
 ER hydroxylation of pro, lys
 Repeating triplets Gly-X-Y (X,Y often Pro, HydroPro),
Y – hydroxy-Lys
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Simple, conjugated proteins
 SIMPLE proteins – albumin, keratin (only AA)
 CONJUGATED proteins:
- Prosthetic group- nonprotein component
- Apoprotein – without prosthetic group
- Holoprotein (apo+ prost)
Types: glycoproteins, metaloproteinss, lipoproteins,
phosphoproteins, hemoproteins
Primary structure and evolution:
Homologues
Conservative, variable
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Tertiary structure, myoglobin, schematic
Mainly alpha helices,
total = 8 helices
(75% of main chain)
Prosthetic (helper)
group to bind O2
Heme group is
protoporphyrin IX,
& central iron atom
TEST
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Distribution of aa in myoglobin
Yellow: hydrophobic aa
Blue: charged aa
White: other aa
Cross-section
Surface, mainly charged aa. Interior, mainly hydrophobic aa
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Porin: “inside out”
Membrane protein
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Protein domains (single polypeptide)
CD4: cell surface protein (immune system), four similar domains
Protein to which HIV attaches
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Quaternary structure, dimer
Cro protein of bacteriophage lambda
Dimer of identical subunits
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Quaternary structure, tetramer
Human
hemoglobin,
two alpha(red)
two beta(yellow)
subunits,
4 heme groups
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Protein denaturation and folding
 Denaturation – loss of 3D structure
 - partially folded state
- Physical condition – T, heat, mechanical stress (foam – egg
white)
- Chemical condition :
- extreme pH (strong acids and bases),
- organic solvents (alcohols, acetone),
- certain solutes – urea, guanidine chloride,
- detergents
- Salt concentration, heavy metals (Pb, Hg-anemia)
- Mild treatment- no covalent bonds
- Need not be equivalent
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Amino acid sequence determines 3D-structure
Bovine ribonuclease, 1950, C. Anfinsen work
4 disulfide bonds
124 amino acids
Denature &
renature
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Reducing disulfied bonds
beta-mercaptoethanol, reduced
oxidized
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Denaturing agent, urea
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Denaturing agent, guanidinium chloride
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Denaturing agent, beta mercaptoethanol
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Ribonuclease: reduction & denaturation
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Reestablishing correct disulfide pairing
Scrambled conformation,
from oxidation in 8 M urea,
only 1% activity,
(105 possible pairings)
Urea removed before
trace of mercaptoethanol
added, full activity
restored.
Process driven by decrease
in free energy
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Transition: folded to unfolded
Sharp
transition
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50 / 50 mixture at halfway
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Patological consequences of
perturbation of protein
conformation
 Prions
 Alzheimer disease
 Beta-Thalassemias
 A prion in the Scrapie form (PrPSc) is an
infectious agent composed of protein in a
misfolded form.
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 A prion is an infectious agent that is composed primarily of
protein. To date, all such agents that have been discovered
propagate by transmitting a mis-folded protein state;
 the protein itself does not self-replicate and the process is
dependent on the presence of the polypeptide in the host
organism.
 The mis-folded form of the prion protein has been implicated in a
number of diseases in a variety of mammals, including bovine
spongiform encephalopathy (BSE, also known as "mad cow
disease") in cattle and Creutzfeldt-Jakob disease (CJD) in
humans.
 All known prion diseases affect the structure of the brain or other
neural tissue, and all are currently untreatable and are always
fatal. In general usage, prion refers to the theoretical unit of
infection. In scientific notation, PrPC refers to the endogenous
form of prion protein (PrP), which is found in a multitude of
tissues, while PrPSc refers to the misfolded form of PrP, that is
responsible for the formation of amyloid plaques and
neurodegeneration.
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Post-translational modification of proteins
•After synthesis of a protein is often attached to the molecule non-protein
component.
Some of the OH group of the side chain (Ser, Thr, ...) is phosphorylated.
Despite nitrogen (Asn) or oxygen (Ser, Thr) is attached oligosaccharide
(glycoproteins)
It is connected acyl fatty acids (lipoprotein) or isotrenová group (anchoring
protein in the membrane)
It is attached prosthetic group required for catalytic function (an organic
molecule, metal ion ...)
Partial proteolysis (insulin, zymogens (pepsinogen, chymotrypsinogen ..., viral
proteins)
Zajímavé linky:
3D modely proteinů
http://www.ncbi.nlm.nih.gov/structure
např. enzym aldolasa
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=69559
Nutné je mít nainstalován plug-in modul Cn3D:
http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml
Aplikace umožňující porovnávat sekvence proteinů
http://www.jalview.org/examples/applets.html
http://www.wehi.edu.au/education/wehitv/
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Finishing touches: covalent modifications
Proteins covalently modified to augment function
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Hydroxyproline
Stabilizes fibers
of collagen in
bone & connective
tissue.
Scurvy: vitamin C
deficiency, leads to
insufficient
hydroxylation
Hydroxylation of proline residues in polypeptide
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gamma-Carboxyglutamate
Carboxylation of glutamate residues in polypeptides
Carboxylation of
glutamate in
prothrombin
(clotting protein)
Vitamin K
deficiency leads to
insufficient
carboxylation, and
hemorrhage
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Carbohydrate to asparagine residues
Addition of sugars
makes proteins
more hydrophilic,
and more interactive
with other proteins
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Phosphorylation of serine, threonine, & tyrosine
Triggered by hormones,
and growth factors.
Phosphorylation is
reversible, thus acts as,
reversible switches for
regulating cellular
processes
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Assisted Folding
 Molecular chaperones- correct folding
 HSP70
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Chaperony