Protein Structure

Question 1

Amino Acids

Answer 1

• joined together by peptide bonds to form the primary structure of the protein
• amino acids have only a few allowed conformations
• the rigidity of the amine bond means that there are only two freely rotating single bonds per amino acid in a peptide backbone
• these bonds are characterised by dihedral angles

Question 2

Ramachandran Plot

Answer 2

• a way of visualising backbone dihedral angles ψ against Ф of amino acids in protein structure
• white areas indicate conformations which are disallowed
• red regions indicate conformations where there are no steric clashes, allowed regions
• yellow regions are areas that are allowed if slightly shorter van der Waals radii are used in the calculation

Question 3

Proteins Secondary Structure

Definition

Answer 3

-produced through intramolecular hydrogen bonding between the protein backbone

Question 4

List the Secondary Structures of Proteins

Answer 4

• alpha helix, between a N-H group and a C=O group
• beta strands and beta sheets
• super-secondary, more complex, structures include beta barrels
• a sequence not forming any secondary structure is said to be intrinsically disordered

Question 5

Proteins Tertiary Structure

Definition

Answer 5

• tertiary structure occurs upon interactions of secondary structure elements
• types of interaction include; hydrogen bonds, ionic bonds, hydrophobic interactions and disulphide bonds

Question 6

Proteins Quaternary Structure

Definition

Answer 6

• occurs in protein containing multiple amino acid chains

- chains are bonded by weak covalent or non-covalent bonds

Question 7

Important Timescales for Proteins in Biological Processes

Answer 7

• atomic oscillations occur at 1fs scales
• protein conformation changes occur at 1ns-1µs scales
• protein folding occurs at 1µs-1s scales

Question 8

Magnitude of Forces Involved in Proteins in Biological Processes

Answer 8

• thermal processes occur at 1fN scales
• hydrogen bond rupture occurs at 1pN scales
• covalent bond rupture occurs at 1nN scales

Question 9

Protein as Bionanomachines

Answer 9

• proteins use mechanical forces in different cell processes
• e.g.
• -translocation
• -activation
• -communication

Question 10

Proteins as Structural Scaffolds

Answer 10

• lamins are α-helical proteins which develop into a network with a lattice-like structure found at the interior of the nuclear envelope
• they provide structural support to the cells nucleus and form an important interface
• the lamin network aids in the coupling of mechanical signals to complex biochemical processes in the cell

Question 11

Polypeptide Chain Collapse and Disease

Answer 11

• homopolypeptide repeats are regions within proteins that comprise a single tract of one particular amino acid
• polyglutmaine chains within the protein Huntingtin are thought to collapse into compact structures, self-assemble and then aggregate to form Lewy bodies (or plaques) within the brain

Question 12

Proteins as Building Blocks for Nanomaterials

Answer 12

-collagen has a hierarchical structure providing it with ability to withstand GPa of pressure and dissipate energy through molecular sliding rather than snapping

Question 13

Proteins as Bionanomacines

Translocation

Answer 13

• translocation is the movement of a protein across a cell membrane
• cellular compartmentalisation requires molecular machinery capable of translocating proteins across cell membranes
• in many cases this first requires protein unfolding
• the translocation rate through a pore depends on the location of the binding to the channel and on the mechanical properties of the protein

Question 14

Proteins as Bionanomachines

Activation

Answer 14

• vWF is a protein that helps blood to clot, it acts like a glue to help stick platelets together
• von WIllebrand disease is a bleeding disorder where the patient wither has low levels of vWF in their blood or their vWF is not working
• shear forces expose a binding site on vWF protein enabling formation of a platelet plug, i.e. activation of the vWF

Question 15

Proteins as Bionanomachines

Communication

Answer 15

• transduction of force between the extracellular matrix and the cytoskeleton is important for cellular function
• single molecule experiments have shown that stretching talin at physiologically relevant forces exposes binding sites
• this site binds with an adhesion protein vinculin leading to cytoskeletal reorganisation

Question 16

Forces Holding Proteins Together

Covalent Bond

Answer 16

• strong bond between two atoms involving sharing of electrons
• range: 0.6-3Å
• energy: ~100-1000kJ/mol

Question 17

Forces Holding Proteins Together

Disulphide Bridges

Answer 17

• covalent bond formed between two reduced -S-H groups
• the strongest bond, can join distant parts of the chain
• range: 2.2Å
• energy: 167kJ/mol

Question 18

Forces Holding Proteins Together

Salt Bridge

Answer 18

• special case of the hydrogen bond
• both donor and acceptor atoms fully charged
• bonding energy is significantly higher than for a hydrogen bond
• range: <3.5Å, typically 2.8Å
• energy: 12.5-17kJ/mol, up to 30kJ/mol for fully/partially buried salt bridges

Question 19

Forces Holding Proteins Together

Hydrogen Bond

Answer 19

• non-covalent interaction between a donor atoms bound to a positively charged hydrogen atom and a negatively charges acceptor atom
• range: <3.5Å, typically 3Å
• energy: 2-6kJ/mol in water, 12.5-21kJ/mol if donor or acceptor is fully charged

Question 20

Forces Holding Proteins Together

Long Range Electrostatic

Answer 20

• non-covalent, coulombic interaction
• between atoms or groups of atoms due to attraction of opposite charges
• depends on the dielectric constant of the medium
• falls of with 1/r, where r is the distance from the atom
• range: variable, 8Å at physiological ionic strength
• energy: depends on distance and environment

Question 21

Forces Holding Proteins Together

van der Waals

Answer 21

• weak attractive force between two atoms or groups of atoms due to fluctuations in electron distributions around nuclei
• stronger between less electronegative atoms e.g. those of hydrophobic groups
• range: 3.5Å
• energy: 4-17kJ/mol, 4kJ/mol on surface, 17kJ/mol on interior

Question 22

How can the potential landscape of a protein be modified?

Answer 22

• thermal (heating and cooling)
• chemical (denaturation)
• mechanical (external forces)

Question 23

Thermodynamic Stability of Proteins

Free Energy, Folding and Unfolding

Answer 23

-assume a transition directly between the folded and unfolded states
-the folded arrangement has a lower free energy than the unfolded
-the transition is reversible
k1 = rate constant for folded to unfolded reaction
k2 = rate constant for unfolded to folded reaction
-rate constants for reactions may be different in each direction
-the equilibrium constant:
keq = k1/k2

Question 24

Thermodynamic Stability of Proteins

ΔGfu and keq

Answer 24

ΔGfu = -kbTln(keq/1M)

• where the 1M is just to normalise the result
• for most proteins ΔGfu is small, ~10-25kbT, roughly equivalent to the energy of a few covalent interactions

Question 25

Thermodynamic Stability of Proteins

Free Energy Equation

Answer 25

ΔGfu = ΔHfu - TΔSfu

• although ΔGfu is typically very small, this can be achieved with both a large enthalpic term, ΔHfu, and a large entropic term, ΔSfu
• however this equation does not match the typical thermodynamic stability curves seen

Question 26

Thermodynamic Stability of Proteins

Modified Gibbs-Helmholtz Equation

Answer 26

ΔGfu(T) = ΔHfu(1+T/Tm) + ΔCp(Tm-T+Tln(T/Tm))

-where Tm is the melting temperature

Question 27

Heat Adapted Organisms

Answer 27

• thermophiles: 55-80’C

- hyperthermophiles: 80-113’C

Question 28

Strategies for Increasing Protein Stability

Answer 28

• shifting the free energy curve to higher temperatures
• broadening the free energy curve
• shifting ΔGfu up at all temperatures

Question 29

Thermostabilisation Strategies

Answer 29

• increased hydrophobic interactions
• increased number of ion pairs and networks of ion pairs
• increase in number of disulphide bonds
• reduced number of unstructured loop regions
• improved packing efficiency
• reduced cavity sizes (more compact)

Question 30

Thermostabilisation Strategies

Conformational Flexibility and Loop Regions

Answer 30

• a decrease in the configurational entropy of the unfolded protein causes a decrease in ΔSfu and therefore an increase in the thermodynamic stability of the protein
• -comparison of sequences of thermophilic and meophilic homologues found that thermophile sequences were shorter usually through shattering of loop regions

Question 31

Themostabilisation Strategies

Increased Hydrophobicity

Answer 31

• increased burial of hydrophobic residues into the interior of the protein is:
• -entropically favourable, it lowers the degree of ordering of water molecules that occurs when side chains are removed from the solvent
• -enthalpically favourable if there is an accompanying increase in van der Waals contributions to the hydrophobicity

Question 32

Thermostabilisation Strategies

Ionic Interactions and Networks

Answer 32

• proteins from thermophilic organisms tend to have a higher percentage of charged amino acids than mesophiles
• salt bridges an ionic bond networks become increasingly important for thermodynamicstability at high temperatures in these proteins