Tertiary and Quaternary Structure of ProteinsLecture 8. 10/15/2014

Reading: Lehninger Ch. 4.3, 4.4

Voet, Voet and Pratt Pg. 148-151; 155-172

Key terms and topics:

Tertiary Structure

Motifs

Domains

Globular proteins

Quaternary Structure

Subunit - monomer v. dimer v. trimer etc.

Protein Folding

Denaturation/Renaturation

Molten globule

Folding Chaperones

PyMOL molecules/PDB files which

contain examples of domains, motifs,

and interesting 3° and 4° structures

Hemoglobin (1THB)

-Hemolysin (7AHL)

Green Fluorescent Protein (1EMA)

Trilobed protease (2GOP)

Triose-phosphate isomerase (2YPI)

Voltage-Dependent Anion Channel – 2JK4

Factors affecting the stability of an -helix

Amino Acid sequence:

Relative side chain position:

Stabilizing effects of the helical dipole:

a helical-wheel projection

Helices that are hydrophilic on one face and hydrophobic

on the other are

An extended zig-zag conformation N

C

-conformation and the -sheet

-turns allow the strand to reverse directionType I contains

Type II contains

N

C

N

C

Finally - a random coil is a region of a protein that doesn’t adopt a 2° structure, though

technically, it is not random - it adopts a conformation defined by the sequence and the

protein which contains it - to reduce the overall free energy.

Random Coil

Circular Dichroism (CD) allows spectroscopic

determination of 2° structure conformation

A scan of wavelength in the far UV region

(where the peptide bond absorbs) is plotted

against (the difference in molar extinction

coefficients of left handed and right handed

plane polarized light).

The tertiary structure of proteins

The components of a tertiary structure: MotifsProtein structure heirarchy:

1° structure } 2° structure } motifs } domains} 3° structures(be careful when talking about these as a hierarchy of structures; an entire protein can be 1 motif,

or 1 motif or more can make up a domain)

Tertiary structure are often represented as ribbon-like ‘cartoons’ that emphasize -helices

and -sheets, (arrows indicate N-term to C-term direction)

-- loop -meander Greek Key -barrel

-coiled coil4-helix bundle

an ‘/’ proteinAn all protein An all protein

Proteins can be broadly classified based on their motifs

Motifs

Large motifs can be constructed from smaller ones:

Beta-sheets

A) have extensive H-bond among main chain atoms.

B) can have parallel and anti-parallel strands.

C) often have alternating hyrdophobic and hydrophilic amino acids.

D) have H-bonds that are roughly perpendicular to the direction of the strand.

E) All of the above are true of beta-sheets.

Clicker Question

Domains in the 3° Structure - An Independently

Stable Portion of the ProteinDomains:

dsRNA binding motif (it is also a domain)

Nuclease domain

Linker

Summary of Factors that Stabilize 3° Structures1) Hydrophobic residues in the protein core.

Exposed hydrophobic side chains forces water to become ordered around them and

decreases the entropy. As these residues are buried in the interior, the water is

released and the entropy increases (energetically favorable).

2) Maximum van der Waals contacts in the core.

“Nature abhors a vacuum” - i.e. gaps in the core will be filled due to the attractive

forces between atoms.

3) Hydrogen bonded 2° structures

Maximized hydrogen bonding and van der Waals contacts in main chain atoms.

4) Amphipathic 2° structures

Increase hydrophobic interactions in the interior; increse H-bonding with water or other

residues on the exterior.

5) Reverse turns

Allow for the peptide chain to reverse direction - requires unusual conformational

flexibility provided by proline and/or glycine and H-bonding.

6) Disulfide bonds

Covalently crosslinks 2 Cys residues that maybe be very far apart in 1° structure but

close in the folded 3° structure.

7) Long range H-bonds - Similar effect as a disulfide, but non-covalent.

Quaternary Structure

If two polypeptides are the same it is a homodimer; if they are different it is a heterodimer.

Dimer (2), trimer (3), tetramer (4), hexamer (6), dodecamer (12). Each monomeric unit is a subunit.

Subunits can have very different functions such as catalysis, regulation, and ligand binding.

Examples: hemoglobin is a tetramer, NDKB is a hexamer

Hb NDKB

How Does a Protein go from String of Amino Acids

to a Beautiful 3-D Structure?

The sequence determines structure:

The Protein Folding ProblemA 100 amino acid protein is synthesized in 5 seconds

Assume one amino acid can have 10 conformations. That’s 10100 conformations/peptide.

Assume a protein folds randomly, trying every possible conformation and each trial

takes 10-13 seconds (the time of a molecular vibration).

…it would take 1077 years to fold a protein.....

Since this is obviously not random a random process, a folding pathway must be built

into the protein (via the sequence).

xkcd.com

Protein Folding Models:

The Stepwise Model:

The Hydrophobic Collapse Model:

Some proteins use ‘Chaperones’ to help them fold

Protein Folding and Disease

Mis-folding can occur, but the proteins are usually degraded

Can lead to certain diseases - Alzheimer’s, Parkinson’s and the prion protein diseases:

Creutzfeldt-Jakob (humans), Bovine spongiform encephalopathy (mad cow), chronic wasting (deer)

Amyloidoses - generation of amyloid fibers - consisting mostly

-sheets - deposited extracellularly. seen as a ‘plaque’

Thought to cause Alzheimer’s. PrP PrPSc

In prions, the mis-folded form

catalyzes the mis-folding of

other prion proteins.

Folding Energetics and Gibbs Free Energy

N

C

When going from unfolded (U) to folded (N) it should be a decrease in free energy, or G,

and we can measure the change in standard free energy when 1 mole of reactants are converted

to 1 mole of products

The G° is dependent on the changes in entropy (S°) and enthalpy (H°) of the system,

related through the Gibbs equation:

G° = H° - TS°

G° = H° - TS°

An energetics review sheet - think about why the following are true:

G is + reaction is unfavorable, requires energy

G is - reaction is favorable, releases energy

H is large G is large

H is small G is small

S is large G is small

S is small G is large

H is - heat is released, bonds are formed

H is + heat is absorbed, bonds are broken

S is large increase in entropy (disorder)

S is small decrease in entropy (disorder)