Lecture 11

– Test next week in class– Protein structure

Collagen• Most abundant protein of vertebrates.• Extracellular protein-insoluble fibers, great tensile strength• Major component of connective tissues (bone, teeth, cartilage,

tendon, ligament, etc.)• Type I collagen-3 polypeptide chains, 285 kD.

– 3000 Å long and 14 Å diameter.

• Distinct amino acid composition; 1/3 are Gly and 15-30% are Pro and 4-hydroxyprolyl (Hyp) residues.

Collagen• Collagen has a triple-helical structure• Amino acid sequence has repeating triplets of Gly-X-Y with X=Pro

and Y= Hyp over 1011 residues out of 1042 residue polypeptide.• Forms a right-handed triple helical structure.

The triple helix of collagen.

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• Shows how left-handed polypeptide helices are twisted together to form a right-handed superhelical structure.

• Individual polypeptide has 3.3 residues per turn and pitch of 10 Å.

• The collagen triple helix has 10 Gly-X-Y units per turn and a pitch of 86.1 Å.

Figure 8-30b X-Ray structure of the triple helical collagen model peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (b)

View along helix axis.

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Figure 8-31 Electron micrograph of collagen fibrils from skin.

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Figure 8-32 Banded appearance of collagen fibrils.P

age

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Figure 8-30c X-Ray structure of the triple helical collagen model peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (c)

A schematic diagram.

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Figure 8-33 A biosynthetic pathway for cross-linking Lys, Hyl, and His side chains

in collagen.

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• Collagen fibrils are covalently cross-linked.

• Collagen almost no cysteine.• It is cross linked y Lys and His.• Lysyl oxidase coverts lysine to

allysine.• Allysine are condesed to

allysine aldol.• This reacts with His to form

Aldol-His.• Aldol-His reacts with 5-hydroxy-

Lys crosss linking the four side chains.

Table 8-3 The Arrangement of Collagen Fibrils in Various

Tissues.

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Globular proteins• Diverse group of proteins that exist as compact spherical

molecules.• Enzymes, transport, and receptor proteins.• Most structural information from X-ray crystal structure

and NMR.• X-ray crystallography directly images molecules.• X-ray wavelengths are small 1.5 Å (visible light is 4000 Å)• X-rays generated by synchrotrons, a type of particle

accelerator to make X-rays of high intensity.

Crystalline proteins• Molecules in protein crystals are arranged in regularly

repeating 3-D lattices.• Unlike other small organic or inorganic molecules,

proteins are highly hydrated (40-60% H2O)• Water is required for the native structure of the proteins.• Generally disordered by >1 Å.• Typical resolution is 1.5 to 3.0 Å.

Figure 8-36a Electron density maps of proteins.

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Figure 8-36b Electron density maps of proteins.

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Figure 8-36c Electron density maps of proteins.

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Figure 8-37 Sections through the electron density map of diketopiperazine calculated at

the indicated resolution levels.

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Crystalline proteins

• Crystalline proteins assume the same structure they have in solution

• Crystals have 40-60% water content (similar to most cells)• Proteins may crystallize in of several forms depending on

conditions. Different crystal forms of the same protein have identical conformations.

• Many enzymes are catalytically active in the crystalline state.

NMR for protein structure determination

• Use of 2D NMR• Yields interaatomic distances between specific protons

that are <5 Å apart.• Interproton distances through space can be determined

by nuclear Overhauser effect spectroscopy (NOESY) • Interproton distance through bonds as determined by

correlated spectroscopy (COSY).• Present methods are good only with molecular masses up

to 40 kD.• Usually well correlated with X-ray data, but sometimes

differs.• NMR can probe motions over time scales of 10 orders of

magnitude so can be used to study protein folding and dynamics.

Figure 8-38a The 2D proton NMR structures of proteins.(a) A NOESY spectrum of a protein presented as a contour plot

with two frequency axes 1 and 2.

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Off diagonal peaks (cross peaks) occur from interaction of 2 protons that are <5 Å apart in space and whose 1D-NMR peaks are located where the horizontal and vertical lines cross through the cross peak intersect the diagonal.Nuclear Overhauser Effect (NOE)

a d

bc

ab

cd

ab

cd

Figure 8-38b The 2D proton NMR structures of proteins.(b) The NMR structure of a 64-residue polypeptide comprising the

Src protein SH3 domain.

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Tertiary structure• Tertiary structure is the three dimensional arrangement of

a protein. • Includes the folding of secondary structural elements and

spatial dispositions of the side chains.• Determined by X-ray crystallography and NMR

Figure 8-39a Representations of the X-ray structure of sperm whale myoglobin. (a) The protein and its bound heme are drawn in

stick form.

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Figure 8-39b Representations of the X-ray structure of sperm whale myoglobin. (b) A diagram in which the protein is represented

by its computer-generated C backbone.

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Figure 8-39c Representations of the X-ray structure of sperm whale myoglobin. (c) A computer-generated cartoon drawing in an

orientation similar to that of Part b.

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Globular proteins have both helices and sheets

• Most proteins have a significant amount of secondary structure

• On average 31% helix, 28% sheet, and a total content of helices, sheets, turns and loops comprising 90% of the structure of a protein.

Figure 8-40 The X-ray structure of jack bean protein concanavalin A.

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Figure 8-41 Human carbonic anhydrase.

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Side chain location varies with polarity• Globular proteins lack the repeating sequences responsiblee for the

regular conformations of fibrous proteins.• The amino acid side chains in globular proteins are distributed

according to polarities.• Nonpolar residues (Val, Leu, Ile, Met, and Phe) occur in the interior of

a protein.• Charged polar residues (Arg, Lys, His, Asp, Glu) are mostly located

on the surface of a protein.• Uncharged polar residues (Ser, Thr, Asn, Gln, Tyr, and Trp) are

usually on the surface but can occur in the interior of the protein. – If in the interior, they are H-bonded to neutralize their polarity.

Figure 8-42a The X-Ray structure of horse heart cytochrome.

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Figure 8-43a The H helix of sperm whale myoglobin. (a) A helical wheel representation in which the side chain positions about the

helix are projected down the helix axis onto a plane.20

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Figure 8-43b The H helix of sperm whale myoglobin.(b) A skeletal model, viewed as in Part a.

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Figure 8-43c The H helix of sperm whale myoglobin.(c) A space-filling model, viewed from the bottom of the page in

Parts a and b and colored as in Part b.

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Figure 8-44 A space-filling model of an antiparallel sheet from concanavalin A.

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Energy diagram of the protein folding process.

The completely unfolded protein is thought to be in the least stable form.For most proteins, the native conformation is the most thermodynamically stable and the only form that is biologically active.

The complete loss of organized structure in a protein is called “denaturation”.

Denaturation results in loss of biological activity!

Denaturation process occurs during cooking an egg.

Denaturants include:• Large evil fire ants??• Heat• Organic solvents• Urea• Detergents• Acid or base• Shear stress• Hydrophobic interfaces

Denaturation and renaturation of a protein

Structural Motifs in Proteins.

• Individual units of 2ndary structure combine into stable, geometrical arrangements.

• Called supersecondary structure or motifs.• Are often repeated in same protein, different proteins.• Certain motifs have associated biological functions:

Helixloophelix motif binds DNA, sequesters calcium ion.

• Secondary structures often depicted as ribbon diagrams• Ribbons invented by Jane Richardson, originally

drawn by hand, now done by computer programs.

Some common structural motifs of folded proteins

a) The motif

(helix-turn helix)

Some common structural motifs of folded proteins

b) The motif; antiparallel

Some common structural motifs of folded proteins

c) The “Greek Key” motif

Some common structural motifs of folded proteins

d) The motif

Several motifs combine to form a superbarrel in the glycolysis enzyme triose phosphate isomerase (TIM barrel)

Quaternary structure• Spatial arrangement of protein subunits.• Polypeptide subunits associate in a geometrically specific manner.• Why subunits?• Easier to repair self-assembling single subunit vs. a large

polypeptide.• Increasing a protein’s size through subunits is more efficient for

specifying the active site.• Provides a structural basis for regulating activity.

Domains in proteins.

• Common sequence regions in native proteins can fold up to form compact structures called “domains”.

• Domains can range in size from 50-400 amino acids, have upper limit in forming compact hydrophobic core.

• Domains are a type of folding motif, typically have separate hydrophobic core.

• Larger proteins are composed of multiple domains, often connected by flexible linker peptide regions.

• Classic example: antibodies

Structural elements of IgGs:

Naturally occurring immunoglobulins (IgG molecules) have identical heavy chains and light chains giving rise to multiple binding sites with identical specificities for antigen.

Antibody Immunoglobulin Domains

Antibodies are composed of: V (for variable) regions - encodes the

antigen binding activityC (for constant) regions - encodes

immune response signal/effector functions:

1. Complement fixation (activation of complement cascade)

2. Binding and activation of Ig receptors (transport from maternal source, activate immune system T cells to engulf, destroy foreign cells, particles, proteins)

3. Also binds bacterial Protein A, Protein G (used in purification)

Note: dashed lines indicate

interchain disulfide bonds

Antibody Immunoglobulin Domains

• There is a conserved glycosylation site in the CH2 domain of IgG (purple region).

• A carbohydrate is covalently attached here by postranslational modification.

Antibody Immunoglobulin Domains

• IgG secondary/tertiary structure: multiple beta-sheet domains.• Termed “immunoglobulin domain”.• Repeated motif in many immune and receptor proteins.

Antibody Immunoglobulin Domains

Modes of Flexibility of IgG structure

Antibody Immunoglobulin Domains

Subunit interactions• Identical subunits in a protein are called protomers• Proteins with identical subunits are oligomers.• Hemoglobin is a dimer (oligomer of two protomers) of

protomers.