Download Protein folding

Preface
[1] Overview of Protein structure
 3-D structure of a protein is determined by its amino
acid sequence (primary structure).
 The function of a protein depends on its structure.
 A protein exists in one or a few stable structural
forms (conformations).
 A protein’s conformation is stabilized largely by weak
interactions.
 Protein structures show common structural patterns
(secondary structures, supersecondary structures,
domains.)
(1) Protein conformation:
- Proteins are not straight lines, but fold into structures almost
immediately after (and during) synthesis (protein folding).
- A protein may assume multiple, thermodynamically stable
conformations (lowest G).
 native conformations
- Proteins undergo conformational change upon ligand binding
or during catalysis.
- Proteins can be denatured (unfolded)
by high temperature (thermal denaturation),
by extreme pH (acid/alkali denaturation),
by denaturants (SDS, urea, etc.)
Folding into a unique conformation
Structure of chymotrypsin
Structure of chalcone synthase from alfalfa
(PDB ID: 1BI5)
Protein Data Bank (PDB)
http://www.rcsb.org/pdb/
Crystal Structures of STS and CHS
Peanut STS
b1d b2d
Alfalfa CHS
b1d b2d
Highly homologous overall structures.
Two b-strands, β1d and β2d at the cyclization pocket, particularly show
discrepancies in amino acid sequences in STS and CHS.
The Nobel Prize in Chemistry for 2008 jointly to
Osamu Shimomura: For the discovery of GFP from jellyfish,
Aequorea victoria in 1962
Martin Chalfie: Use of GFP as a luminous genetic tag
for various biological phenomena
Glowing marker allows us to watch
the movements, positions and interactions of
the tagged proteins
Roger Y. Tsien: Mechanism of GFP fluorescence
Engineering different GFP mutants with
different colours
Aequorea victoria
www.bbc.co.uk
srv2.lycoming.edu
GFP drawn in cartoon style, once fully and
once with the side of the beta barrel cut away to reveal
the fluorophore (highlighted as ball-and-stick).
en.wikipedia.org
FSYGVQ
Cody, C. W. et al., Biochemistry (1993) 32, 1212-18
Heim, R., Prasher, D. C., Tsien, R. Y.,
Proc. Natl. Acad. Sci. USA (1994) 91, 12501-04
Förster cycle
http://www.cryst.bbk.ac.uk/PPS2/projects/jonda/chromoph.htm
The diversity of genetic mutations:
living bacteria expressing 8 different colors of fluorescent proteins.
en.wikipedia.org
Immunofluorescence of EYFP-CVLL CHO-K1 cells and native CHO-K1 cells.
Panel A: EYFP-CVLL cell, 10 h after seeding;
Panel D: EYFP-CVLL cells was treated with 20 M lovastatin for 24 h
Liu, X.-h., Suh, D.-Y., Call, J., Prestwich, G. D. (2004) Bioconjugate Chem. 15, 270-277
Genetically-engineered zebra fish:
luminating beauties with practical applications
GloFish: Zebra Fish as Pollution Indicators
http://www.nus.edu.sg/corporate/research/gallery/research12.htm
Green-glowing pigs
Red Fluorescent Cat Cloned
http://news.bbc.co.uk/1/hi/world/asia-pacific/4605202.stm
http://upload.wikimedia.org/wikipedia/commons/e/ec/Cloning_diagram_english.svg
(2) Protein conformations are stabilized by
Disulfide bonds (~210 kJ/mol),
weak (4~30 kJ/mol) noncovalent interactions
(H-bonds, ionic and hydrophobic interactions, van der Waals
forces).
What drives the protein folding?
 Hydrophobic interactions: hydrophobic residues are largely
buried in the protein interior.
 H-bonds: the number of H-bonds within the protein is
maximized.
 Ionic interactions and van der Waals forces also contribute.
(3) Peptide Bond: the linkage between two amino acids
- Condensation reaction: -COOH + -NH2
water is eliminated
(amide linkage or amide bond)
-C-NH- + H2O
O
- The peptide C‒N is shorter than an amine’s C‒N bond.
This indicates a resonance, or sharing of electrons.
 partial (40%) double bond character
- The six atoms of the peptide group is coplanar.
- O of the carboxyl and H of the N-H are in
trans configuration
Electric dipole
Free rotation
No rotation about the peptide bond.
 Limited range of conformations.
- Free rotation about the N(H)‒C bond:  (phi)
Free rotation about the C‒C(=O) bond:  (psi)
-  and  = 180o when polypeptide is in its fully extended
conformation and all peptide bonds are in the same plane.
- +180o >  and  > ‒180o
Due to the size and charge of the R groups,  and  are
rotationally hindered.
 Ramachandran Plot
-
(http://www.cgl.ucsf.edu/home/glasfeld/tutorial/AAA/AAA.html)
( =  = 0o not allowed.)
b
No steric overlap
Allowed at the
extreme limits

Not
allowed.
(4) Secondary (2°) structure:
 The general three-dimensional form of local segments of
proteins (or nucleic acids).
 Defined by patterns of hydrogen bonds between
backbone amide groups.
(side chain-main chain and side chain-side chain
hydrogen bonds are irrelevant).
 The most prominent are -helices and b-sheets
(b-strands).
(a) -Helix
 polypeptide backbone is wound around
imaginary axis with R group protruding outwards.
 3.6 amino acid residues
per helical turn.
 The carbonyl group (-C=O)
of each peptide bond
extends parallel to the
axis of the helix and
points directly at the
-N-H group of the peptide
bond 4 amino acids
below it in the helix.
(a) -Helix
 Stabilized by H-bonds between C=O(n) and
N-H(n+4).
[‒N-H·····O=C‒]
R groups protrude outwards
Top view
 Only extended right-handed
-helices are found
in proteins.
 Extended left-handed helices
are rare in proteins.
31 verified left-handed helices in a
set of 7284 proteins.
Short: < 6 a.a.
Rare, but when they do occur, they
are structurally or functionally
significant.
Right-handed
Mirror images
 Certain amino acids destabilize a helix
1. D-amino acids
2. long blocks of Glu, Lys, Arg
3. bulky side chains such as Asn, Ser, Thr, Leu
(if close together in sequence).
4. Pro and Gly (helix breakers)
 Favourable interactions between R(n) and R(n+3 or 4)
stabilize the helix.
① +ve and ‒ve (ion pair)
② Two aromatic/hydrophobic residues
(-stacking/hydrophobic interactions)
③ ‒ve a.a. at the N-end & +ve a.a. at the C-end
due to the helix dipole.
The tendency of a given segment of a
polypeptide chain to fold up as an  helix
depends on the identity and sequence of
amino acid residues within the segment.
⇒ Sequence determines the structure.
(b) b-Sheet: polypetide chain is extended in a zigzag fashion.
• has hydrogen bonding as in -helices (all C=O and N-H),
but between chains rather than within one chain.
• R groups of adjacent amino acids protrude in opposite
directions (up, down, up,….)
• b-sheets come in two varieties: parallel and antiparallel
C-end
N-end
C-end
N-end
 Small R groups are preferred when b-sheets are layered
together within a protein/
 High Gly and Ala contents in b-keratins (silk and spider web).
Between parallel and antiparellel b–strands,
which one is more stable?
(c) b-Turns: reverse directions in compact globular proteins.
 Gly and Pro preferred
 b-turns, -turn, and cis-Pro
(d) Secondary Structure Propensities of Amino Acids
Secondary structure prediction
⇒ Chow‒Fasman Method
[3] Protein Tertiary and Quaternary Structures
Tertiary (3°) structure:
3-D arrangement of all atoms in a protein.
3-D folding of the secondary structural elements.
Forces that influence folding
(usually all are optimized):
1. Hydrogen bonding
2. Hydrophobic interactions
3. Ionic interactions
4. Van der Waals forces
5. Disulfide bridges
6. Metal chelation
(cross linking)
(1) Fibrous proteins:
 Polypeptide chains are arranged in lone strands or
sheets
 2o structures:
A single type (helix or sheets)
Simple repetitive pattern
 Roles
Structural supports, protection
-keratin, collagen, silk fibroin (Table 4-1)
(a) -Keratin and Hair
Box 4-2: Permanent Waving
(b) Collagen and Tendons, Cartilage
Gly-X-Pro (4-Hyp) repeating units

Left handed helix (3 a.a./turn)

Triple helix (superhelix) of 3 helices

X-linking in collagen fibrils (aging)
End-view of collagen superhelix
Gly in red.
Collagen fibrils
A GlySer(Cys) mutation in -chain
 Osteogenesis imperfecta
Ehlers-Danlos syndrome
4-Hyp and Scurvy (Vitamin C deficiency)
Box 4-3, p. 130
Eat your fresh fruits and vegetables.
Prolyl-4-hydroxylase
(c) Silk Fibroin
Extended b-conformation
Rich in Ala and Gly, allowing close packing
Man-made spider silk
Silk proteins expressed in (transgenic) goat milk
($20,000/L)
Biodegradable fishing line
Micro suture
Ballistic armour
Nexia Biotechnologies (Canadian) & Pentagon
http://www.azom.com/details.asp?ArticleID=1543
(2) Globular Proteins
 Compactly folded
 Structural diversity  Functional diversity
 Finite folding patterns
(a) Myoglobin:
153 a.a. residues, O2 carrier, Heme as a prosthetic group.
Rich in diving mammals.
Eight -helices connected by bends (b-turns).
# Stabilized by hydrophobic interactions (and vdW forces).
⇒ Most hydrophobic residues oriented toward the interior of the
molecule (Hydrophobic core)
# Compact ⇒ Only 4 H2O molecules can fit inside.
# Polar R groups on the surface ⇒ Solubility
# Buried polar groups have H-bond partners.
# All -helices are right handed.
 3 Pro found at bends & 1 Pro within an -helix, causing a kink
necessary for tight packing.
 Heme (Fe2+) is bound (coordination bond) to His93, well
shielded from outside.
#Common features in all proteins.
(b) Structure Diversity
(3) Common Structural Patterns:
Supersecondary structures & Domains
 There are unlimited possibilities for protein tertiary
structures, yet there are common structural patterns that
recur in different and often unrelated proteins.
⇒Supersecondary structures (Motifs, Folds):
A distinct, stable folding patterns for elements of secondary
structure
Smaller motifs can contribute to larger ones.
The b--bloop (left) contributes to
the /b barrel (right).
4-Helix bundle:
The four-helix bundle is found in a number of
different proteins. In many cases the helices
part of a single polypeptide chain, connected
to each other by three loops.
β barrel (Greek Key β-sheets)
This motif is made of 4 strands of an antiparallel β-sheet linked by
short connections (hairpins) between strands a, b, and c. A longer
loop between strands c and d allows a and d to form hydrogen
bonds. This motif is named based on its similarity to the pattern
seen on Greek pottery. This motif occurs frequently in β-barrel
structures.
α/β barrel (TIM barrel)
The eight-stranded /b barrel (TIM barrel, named after triose phosphate
isomerase) is by far the most common tertiary fold. It is estimated that 10% of
all known enzymes have this supersecondary structure. The members of this
large family of proteins catalyze very different reactions. Currently, there are
85 enzymes in the TIM database including oxido/reductases, hydrolases,
dehydrogenases, isomerases, structural proteins, etc.
• Domains are often a stable portion of 3° structure
• Often made from two or more covalently linked motifs
• Proteins can consist of one or more domains linked as one
polypeptide unit, each being structurally independent with
the characteristics of a small globular protein and
functionally distinct (DNA bind domain, cytosolic domain of
membrane proteins, catalytic and regulatory domains, etc)
Separate Ca2+ binding
domains in
polypeptide troponin C
(4) Structural Classification of Proteins (SCOP)
http://scop.mrc-lmb.cam.ac.uk/scop
Structural hierarchy: ClassFoldSuperfamilyFamily
 Finite folding patterns  3o structure is more conserved than
primary sequence
 Each fold further divided based on evolutionary relationships
Superfamily: Made of several families.
Little sequence similarity, share motifs and functional
similarities.
 Probable evolutionary relationship
Family: Sequence similarity and/or similar structure/function.
 Strong evolutionary relationship
Classes:
1. All alpha proteins [46456] (218) [unique ID no.] (no. of folds)
2. All beta proteins [48724] (144)
3. Alpha and beta proteins (a/b) [51349] (136)
Mainly parallel beta sheets (beta-alpha-beta units)
4. Alpha and beta proteins (a+b) [53931] (279)
Mainly antiparallel beta sheets (segregated alpha and beta
regions)
5. Multi-domain proteins (alpha and beta) [56572] (46)
Folds consisting of two or more domains belonging to different
classes
6. Membrane and cell surface proteins and peptides [56835] (47)
Does not include proteins in the immune system
7. Small proteins [56992] (75)
Usually dominated by metal ligand, heme, and/or disulfide
bridges
SCOP Classification Statistics
SCOP: Structural Classification of Proteins. 1.69 release
25973 PDB Entries (July 2005). 70859 Domains.
Class
Number of
folds
Number of
superfamilies
Number of
families
All alpha proteins
218
376
608
All beta proteins
144
290
560
Alpha and beta
proteins (a/b)
136
222
629
Alpha and beta
proteins (a+b)
279
409
717
Multi-domain proteins
46
46
61
Membrane and cell
surface proteins
47
88
99
Small proteins
75
108
171
Total
945
1539
2845
(5) Protein Quaternary Structure of Multisubunit Proteins
 Most proteins larger than 100 kDa consist of more than one
polypeptide
 Multimer, Oligomer, Protomer (repeating structural unit)
 The tertiary structures of each chain will associate together
with a specific geometry
 The spatial arrangement of the subunits is called the
quaternary structure
Hemoglobin,
2b2 tetramer
(dimer of b
protomers)
Viral capsids
Jellyfish Aequorea victoria
(6) Protein Denaturation and Folding
(a) Denaturation:
Loss of 3-D structure sufficient
to cause loss of function
 Unfolding is abrupt and
a cooperative process.
 Tm: melting point
 Denaturants: heat, pH,
organic solvents, detergents
urea, guanidine chloride (GdnHCl)
O
H2N C NH 2
NH2 Cl
H2N C NH2
(b) Unfolding and
Renaturation (refolding)
Anfinsen’s Experiment:
Spontaneous folding of
denatured ribonuclease
The first proof for
“sequence determines structure”
What would happen if the
denatured enzyme is first
oxidized to form S-S bonds
and then the urea is
removed ?
(c) Protein folding
 Fast process:
In E. coli, a 100 a.a. long protein folds into a native
conformation in ~5 sec at 37oC.
Levinthal’s paradox: A random process through all possible
conformations will take ~1077 yrs.
 Folding process is not random.
Then, how? Two possible models.
① Hierarchic process in which local 2o structures form first,
followed by longer-tange interactions between those local
2o structures to form domains and the native structure.
② “Molten globule” by a spontaneous hydrophobic collapse
that folds into the native structure.
 Real process may follow both models.
Computer-simulated folding pathway
of a 36-residues protein.
(c) Protein folding
 Thermodynamics of protein folding depicted
as a free-energy funnel.
 Protein molecules have
flexible regions with low stability
that bring about
conformational change,
essential to function.
(d) Misfolding and diseases (The prion diseases)
Mad cow disease (BSE, bovine spongiform encephalopathy)
Kuru, Creutzfeldt-Jacob disease, scrapie,..
 Prion (PrP) is the causative agent
(the first and only such protein).
 PrP exists in two conformations,
PrPC - normal form (42% -helices, soluble),
its function is inessential for life.
PrPSc- altered conformation (43% b-sheets, insoluble,
forms neurotoxic aggregates).
 PrPSc converts PrPC to PrPSc in chain reaction.
 transmissible by contact with just the PrPSc form.
 Inherited forms of prion diseases caused by
mutations in the PrP gene, including LeuPro and
AspAsn point mutations.
PrPC
PrPSc