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3D Structure of Proteins

Biochemistry Lecture
by Heidi Fletcher on 31 August 2014

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Transcript of 3D Structure of Proteins

3D Structure of Proteins
Levels of structural
organization

Primary
1(knot)

AA seq.
Determined
by chemical
tests

Secondary
2(knot)

Local,
regular folding

Characteristics
Folding stabilized
by H-bonding

Bond
Lengths

Interatomic
Distance

Amide groups
Planar and trans

3 Types
alpha-helix
Characteristics
& Descriptions

Types
Examples
Fibrous
proteins

Alpha-
Keratins

Collagen
(triple-helix)

Beta-sheets
Characteristics
Beta-
bulge

Practice
Drawing
them out

Parallel vs. Anti-parallel
Turns
Reverse
Turns

Nonrepetitive
structure

Omega loops
"The information for determining the three-
dimensional structure of a protein is carried
entirely in the amino acid sequence of that
protein."
Voet and Voet
“The properties of a protein are determined
largely by its three-dimensional structure.”
Many conformations are possible for proteins:
Due to flexibility of amino acids linked by peptide bonds


• At least one major conformations has biological activity, and hence is considered the protein’s native conformation
Protein Structure
Structure derived criteria for probable conformations
Bond lengths compatible with small peptide structures
Interatomic distances not less than van der Waals radii
Amide group (peptide bond) is planar and in trans configuration. Rotation only about bonds adjacent to Ca.
Folding stabilized by hydrogen bonding (amide protons and carbonyl oxygens)
Secondary Structure
Standard bond distances in Angstroms (Å). The amide bond is planar and the adjacent sidechains are trans.
trans cis
+
C
O
Secondary structure
The fully extended conformation corresponds to  =  = 180° (in trans conformation) has  and  values of 180º

Conformational freedom of a polypeptide backbone is constrained due to steric clashes between amide H, carbonyl O, and C-R

These can be close together in sequence or far apart
The backbone of a protein is a linked sequence of rigid planar peptide groups
Thus, the torsion angles about the C-N bond () and the C-C bond () of each AA can be used to specify the polypeptide’s backbone conformation
Torsion Angles ( and )
Proline is much more likely to form cis peptide bonds than any other residue (~10% of Pro bonds are in cis conformation)
Peptide bond in i-1 position to Pro doesn't have double bond characteristic
Steric interference between adjacent residues
Helices can be left (negative n)- or right (positive n)-handed b/c they are chiral. Only stable helices are formed in proteins. Helices are significantly stabilized by H-bonding. (n = 2 = nonchiral ribbon)
If a polypeptide chain is twisted by the same amount about its C atom, it assumes a helical conformation
A helix can be characterized by the number, n, the number of peptide units per helical turn and by its pitch, p, the distance that the helix rises along its axis per turn
Helical Structures
The  helix is the only helical conformation that has both, allowed torsion angles and a favorable H-bonding pattern
3.6 residues per turn; translation of 1.5 Å per residue (pitch of 5.41 Å per turn)
Average length of ~12 residues per helix in proteins
Side chains project outward and down; core is tightly packed
The N-H bond of residue n points along the helix towards the peptide C=O group to form a H-bond with the (n-4)th residue
1st 4 N-H and last 4 C=O do not participate in H-bonding
Pro is a “helix breaker”, but fits well at N-terminus of helix; also electrostatic repulsion by charged residues and bulky residues
Isolated helices are fairly unstable
The a helix
-Helix (Cont’d)
R groups project downward and outward from the helix so as to avoid steric interference with the polypeptide backbone
Stereo, space-filling representation of an a helical segment of sperm whale myoglobin (its E. helix) as determined by X-ray crystal structure analysis.
In -sheets the polypeptide is in its most extended conformation
Unlike  -helix, H-bonding occurs between neighboring chains instead of within one
Average length of 6 residues/strand and 2-12 strands /sheet
Antiparallel sheets are thought to be more stable than parallel sheets-WHY?
Side-chains from adjacent residues on a strand protrude from opposite sides of sheet (7Å between R groups)
-sheet
Out of plane
In plane
-sheets have a pleated appearance, which accounts for the commonly used “ -pleated sheet” name

Notice R groups of each chain alternately point to opposite sides of the chain

-sheets in proteins are not planar and flat, but have a right handed-twist due to interactions between residues in extended chain

Strands can be connected in several ways:
A two-stranded b antiparallel pleated sheet drawn to emphasize its pleated appearance
Regular 2° structures (helices & sheets) comprise about half of the average globular proteins. The rest of the polypeptide is found to have coil or loop conformations that are irregular and difficult to describe
Stereo, space-filling representation of a six-stranded antiparallel  -sheet in jack bean concanavalin A as determined by x-ray crystal structure analysis.
-Pleated Sheet (Cont’d)
-bulge- a common nonrepetive irregular 2˚ motif in anti-parallel structure
Reverse turns ( bends) - usually defined by four residues and stabilized by intrachain H-bonds between residues 1 and 4

Type 1
Type 2 ( residue 3 usually glycine)

Proline often occurs at residue 2 in both types
Often join 2° structure elements
Best characterized turns are -hairpins (-turns)
Type I and II differ by 180° flip of peptide bond between residues 2 and 3
2 residues involved in H-bond
Reverse turn (or -bend/-turn)
Ω loop comprising residues 40 to 54 of cytochrome c
An Ω -loop is made of 6-16 residues that are not components of helices or -sheets and whose end-to-end distance is less than 10 Å. The side chains fill the interior of the loop.
Ω loops: 6-16 residue segments with end-to-end distances within ~1.0 nm
Importance of turns and loops
Loop regions often exposed to solvent; they can be highly charged.
Very often substitutions between related proteins occur in loop regions.
Loops and turns can form binding sites (esp. antigen binding sites)
Nonrepetitive structure
The peptide group has a rigid and planar structure

Peptide bonds almost always assume the trans conformations due to steric interference
Tertiary
3(knot)

Determined by:
1. x-ray crystallography
2. 2D NMR
Compact
arrangement
of locally folded chains
Globular proteins
can contain both
alpha and Beta
structures
Example
Define
Characteristics
Sperm whale
myoglobin
Residue occurence
Result from the
HYDROPHOBIC
EFFECT

Structural
folding motifs/domains
Domains
aka
Supersecondary
structures
Beta alpha Beta
Beta hairpin
Alpha Alpha
Greek Key
Saddle and Barrel
Beta Sheet
Quaternary
4(knot)

Compact
arrangement
of multiple
peptide chains
Forms supermolecular
structure
Usually has symmetrical subunits

(Hydrophobic effect:
nonpolar residues @ interior of protein
polar residues @ exterior of protein)
This structure provides:

1. Stability
2. Possibility for cooperativity
3. Mulitple catalytic sites
w/in a small volume/area
Protein Stability
Define
Delta G = Delta H - T(Delta S)
Determinants
for stability

H-bonding
Electrostatic
interactions

van der Waals
forces

Hydrophobic
Effect

contributes little
important
*Most significant
Scale
plot
Driven by
Delta S (entropy),
not Delta H (enthalpy)

Denaturnation
of proteins

How?
Renaturation
Protein folding
Example:
Ribonuclease A

Conclusion
from study

A protein's
primary structure
determines
its 3D structure
and thus function

Forces in
tertiary structures:

Covalent
interactions

Noncovalent
interactions

H-bonding
Hydrophobic
Electrostatic
attractions/repulsions
Pathways
Native
conformation
Levinthal
Take-home
message
Examples:
BPTI
PDI
Not a random process!
Example of hierarchial
of protein folding
1. Rapid local
secondary structure
folding (msec)
2. Forms molten globule
3. Next 5-1000 ms,
secondary structure
stabilized and tertiary
structure forms = subdomains
4. Next few sec, side chain
packing occurs when water
is squeezed out of
hydrophobic core = native state reached
Landscape Theory of
Protein Folding
Follows a direct pathway...NOT RANDOM!
4 possibilities
Ideal funnel
landscape
Levinthal
golf course
landscape
Classical
funnel
landscape
Rugged
landscape*
Molecular Chaperones
Fibrous proteins: contain polypeptide chains organized approximately parallel along a single axis. They
consist of long fibers or large sheets
tend to be mechanically strong
are insoluble in water and dilute salt solutions
play important structural roles in nature
Examples are
keratin of hair and wool
collagen of connective tissue of animals including cartilage, bones, teeth, skin, and blood vessels
Fibrous Proteins
Fibrous proteins are highly elongated molecules whose secondary structures are their dominant structural motifs
Often play structural roles (skin, tendon, bone) or have motive functions (muscle and ciliary proteins)

Keratin
Keratin is a mechanically durable and chemically stable protein found in the outer epidermal layer, in hair, horn, nails, & feathers
“hard”  keratin is rich is Cys (many disulfides); giving it extra strength; “soft”  keratin has less sulfur and is more playable
Fibrous Proteins
A typical hair is 20 µm in diameter and is made from dead cells packed with macrofibrils
The macrofibril is made up of microfibril, which is composed of 4 protofibrils
Each protofibrils arises from the dimerization of 2 protofilament.
Protofilaments are formed by the association of keratin coiled-coils in a staggered and antiparallel manner
The central 310 residues of Type I and II  keratin associate to form a coiled coil
Hair is composed mainly of  keratin
a-Keratins - a helix predominant
basic structure is a dimer of helices coiled, which in turn associate to form dimeric “protofilaments.” (coiled coils)
Cysteine rich with interchain
(-S-S-) bridges
extensible
Fibrous Proteins
There is a 5.1 Å pitch due to the tilting of the axis. This inclination allows the side chains to interlock and stabilize the structure.

The position of the residues around the coiled coil are designated the letters a to g.

a and d are usually hydrophobic

e and g are usually charged
Coiled-coils are formed when 2 or 3 helices wrap around one another to form a left-handed “superhelix”
Coiled Coil
Post translational modifications are performed by prolyl 4-hydroxylase or lysyl 5-hydroxylase; both require ascorbic acid (vitamin C)

Scurvy results from lack of vitamin C, which leads to decreased Hyp production, resulting in weak blood vessels and death
Major component of bone, teeth, cartilage, tendon, skin, and blood vessels.
Very repetitive sequence: (-Gly-X-Y-) where X is often Pro and Y is often 4-hydroxyproline (Hyp) or 5-hydroxylysine (Hyl) (non-standard AA’s)
Collagen (Triple Helix)
Pro prevents the protein from forming a standard alpha-helix, instead forming a left-handed helix (~3 residue per turn). Three collagen units form a slightly staggered right-handed superhelix

Every 3rd residue comes into very close proximity to the other two chains; therefore only Gly is allowed

NH of Gly H-bonds with C=O of neighboring X in another collagen molecule within the same superhelix

Pro imparts rigidity to the superhelix structure due to its conformational inflexibility. 4-hydroxy Pro stabilizes the helix by introducing additional H-bonding, while 5-hydroxy Lys allows for cross-linking and attachments of sugar groups.
Collagen (Triple Helix)
Osteogenesis imperfecta (brittle bone disease)
Ehlers-Danlos syndromes
Osteoarthritis
Atherosclerotic plaques
What diseases can be caused by collagen defects?
Collagen is organized into fibrils
X-ray crystallography
uses a perfect crystal; that is, one in which all individual protein molecules have the same 3D structure and orientation
exposure to a beam of x-rays gives a series diffraction patterns
information on molecular coordinates is extracted by a mathematical analysis called a Fourier series
2-D Nuclear magnetic resonance
can be done on protein samples in aqueous solution
Determination of 3° Structure
X-Ray and NMR Data
Determines solution structure
Structural info. Gained from determining distances between nuclei that aid in structure determination
High resolution method to determine 3˚ structure of proteins (from crystal)
Diffraction pattern produced by electrons scattering X-rays
Series of patterns taken at different angles gives structural information
Protein tertiary structure is the 3D
arrangement of the secondary structural
elements, including the spatial disposition of
the side chains.

Globular proteins, so named from their shape, may
contain both a and b structures. The protein
interior is typically non-polar and is efficiently
packed.
Tertiary Structure
Globular proteins: proteins which are folded to a more or less spherical shape they tend to
be soluble in water and salt solutions
most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and ion-dipole interactions
most of their nonpolar side chains are buried inside
nearly all have substantial sections of -helix and -sheet
Globular Proteins
Sperm Whale Myoglobin
153 residues (121 comprise 8 helices)
helices labeled in alphabetical order (residues bear a helix label as well as sequence label, ex. F8)
Compact globule (4.4 x 4.4 x 2.5 nm)
Heme prosthetic group provides oxygen binding function (hydrophobic pocket).
Structure of Myoglobin (Mb)
F8
E7
F
E
D
C
H
B
A
Model of Myoglobin
Nonpolar residues Val, Leu, Ile, Met, and Phe are generally associated with the protein interior.
Charged residues Arg, Lys, Asp and Glu are usually surface deployed.
Uncharged polar groups Ser, Thr, Asn, Gln, Tyr, Trp and His often occur at the surface but if buried, form H-bonds.
Residue Occurrence
Protein tertiary structure is comprised of one or more individual domains of locally folded regions of secondary structural elements.

Domains are linked by coils or loops of polypeptide strand.

Larger globular proteins display multiple domains, whereas small proteins (myoglobin) may have only one domain.

Prototypical protein: 31% a-helix and 28% b-sheet.
Structural Folding Motifs
Structurally independent units, typically connected by a flexible segment of polypeptide chain.

Structural and functional characteristics of a globular protein

100-200 residues with a diameter of ~ 2.5 nm

Often comprised of multiple layers of secondary structure

Domains often have specific function
Domains
One subunit of glyceraldehyde-3-phosphate dehydrogenase
bab motif – a-helical linker between two parallel b strands

Usually this motif contains a right-handed crossover and is the most common form of supersecondary structure

The bab motif is common for proteins that contain a/b domain structures
bab Motif
hairpin motif (aka b-meander):
Antiparallel b-sheet formed by
sequential segments of
polypeptide chain that are linked
by tight reverse turns (hairpin)
b Hairpin
Antiparallel b sheets often assume an interlocking arrangement of strands that is suggestive of decorative patterns observed on classical Greek vases
This Greek Key motif is observed in the concanavalin A structure (residues 146-220)
Greek Key
aa (or helix-turn-helix) motif is characterized by two successive antiparallel -helices linked by a short turn

The helices associate to maximize sidechain interactions

Axes are commonly inclined at a 15° angle which allows accommodation of the sidechains for the two helices
Helix-Turn-Helix
X-Ray structures of 4-helix bundle proteins: E. coli cytochrome b562
A common topology observed for parallel b-sheet structures is the eight-stranded barrel

The strands are linked by right-handed crossovers typically incorporating helices (the bab element)

Hydrophobic residues are sequestered in the interface between the two concentric cylinders
Barrel
Parallel b Sheet Topology
Another common topology for b sheets is the doubly wound parallel b sheet (saddle)
The saddle topology exhibits three layers of peptide backbone and thus has two regions rich in hydrophobic residues
Parallel b Sheet Topology
Antiparallel b sheets can form double sheet, double layered structures with one face (polar side chains) of each sheet exposed to solvent and the other (hydrophobic) face interacting
An example of this b sandwich is the immuno-
globulin fold
Antiparallel b Sheet Topology
Antiparallel b sheets of greater than 6 strands can form single sheet barrel structures
An example of this is the 8 strand up - and - down b barrel as observed in the retinol binding protein
Antiparallel b Sheet Topology
“Native proteins are marginally stable entities under
physiological conditions…protein structure is the
result of a delicate balance among powerful counter-
vailing forces.”

Protein structure and stability is subject to thermodynamics

DG = DH - TDS

Conformational free energy destabilizes protein folding
Protein Stability
The Kyte-Doolitle Hydropathy Index (J. Mol Biol. 157, 110 (1982)) ranks the amino acid side chain hydropathies. This scale can be used as a predictor for the location of a polypeptide chain segment, whether buried or surface exposed.
Ile 4.5 Gly -0.4 Glu -3.5
Val 4.2 Thr -0.7 Gln -3.5
Leu 3.8 Ser -0.8 Asp -3.5
Phe 2.8 Trp -0.9 Asn -3.5
Cys 2.5 Tyr -1.3 Lys -3.9
Met 1.9 Pro -1.6 Arg -4.5
Ala 1.8 His -3.2
Hydropathy Scale
Nonpolar groups are usually buried in the interior of the protein








Hydropathic index plot for bovine chymotrypsinogen. Area above the horizontal line denotes the interior of the protein.
Hydropathy plot
1. The solvation of nonpolar molecules by water molecules results in a decrease in entropy. This decrease in entropy results from the ordering of water molecules around the nonpolar molecule to recover the H-bonds that were lost because of the presence of the hydrophobic molecule.
2. The aggregation of the nonpolar molecules thereby minimizes the surface area of the cavity, and therefore, the entropy loss of the entire system.
The hydrophobic effect is driven by entropy not enthalpy
Subunit interaction:
contact regions resemble protein interior
in that the nonpolar residues are predominant
Dynamic
stimulations
Practice problems
Chapter 6 (GG):
3, 4, 7, 8, 11, 12

Chaperonins
Lecture Notes...
The hydrophobic effect is the entropy-driven tendency of water to minimize its contact with nonpolar substances.

The hydrophobic effect causes nonpolar substances to aggregate resulting in a structure that has lower surface area than the sum of the non-aggregated surfaces area.
Decreased surface area contact between nonpolar substances and water minimizes water's need to organize around the nonpolar substance resulting in a net entropy.

The hydrophobic effect is a poor choice for the name since the largest contribution to the favorable delta G is entropy, whereas hydrophobic effect implies that enthaply between hydrophobic groups is important.

Hemoglobin
Values that define relative hydrophobicity of AA residues; the more positive the value the more hydrophobic
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