Protein Structure Flashcards
1
Q
Amino Acids
A
- 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
2
Q
Ramachandran Plot
A
- 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
3
Q
Proteins Secondary Structure
Definition
A
-produced through intramolecular hydrogen bonding between the protein backbone
4
Q
List the Secondary Structures of Proteins
A
- 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
5
Q
Proteins Tertiary Structure
Definition
A
- tertiary structure occurs upon interactions of secondary structure elements
- types of interaction include; hydrogen bonds, ionic bonds, hydrophobic interactions and disulphide bonds
6
Q
Proteins Quaternary Structure
Definition
A
- occurs in protein containing multiple amino acid chains
- chains are bonded by weak covalent or non-covalent bonds
7
Q
Important Timescales for Proteins in Biological Processes
A
- atomic oscillations occur at 1fs scales
- protein conformation changes occur at 1ns-1µs scales
- protein folding occurs at 1µs-1s scales
8
Q
Magnitude of Forces Involved in Proteins in Biological Processes
A
- thermal processes occur at 1fN scales
- hydrogen bond rupture occurs at 1pN scales
- covalent bond rupture occurs at 1nN scales
9
Q
Protein as Bionanomachines
A
- proteins use mechanical forces in different cell processes
- e.g.
- -translocation
- -activation
- -communication
10
Q
Proteins as Structural Scaffolds
A
- 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
11
Q
Polypeptide Chain Collapse and Disease
A
- 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
12
Q
Proteins as Building Blocks for Nanomaterials
A
-collagen has a hierarchical structure providing it with ability to withstand GPa of pressure and dissipate energy through molecular sliding rather than snapping
13
Q
Proteins as Bionanomacines
Translocation
A
- 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
14
Q
Proteins as Bionanomachines
Activation
A
- 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
15
Q
Proteins as Bionanomachines
Communication
A
- 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
16
Q
Forces Holding Proteins Together
Covalent Bond
A
- strong bond between two atoms involving sharing of electrons
- range: 0.6-3Å
- energy: ~100-1000kJ/mol
17
Q
Forces Holding Proteins Together
Disulphide Bridges
A
- 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
18
Q
Forces Holding Proteins Together
Salt Bridge
A
- 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
19
Q
Forces Holding Proteins Together
Hydrogen Bond
A
- 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
20
Q
Forces Holding Proteins Together
Long Range Electrostatic
A
- 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
21
Q
Forces Holding Proteins Together
van der Waals
A
- 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
22
Q
How can the potential landscape of a protein be modified?
A
- thermal (heating and cooling)
- chemical (denaturation)
- mechanical (external forces)
23
Q
Thermodynamic Stability of Proteins
Free Energy, Folding and Unfolding
A
-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
24
Q
Thermodynamic Stability of Proteins
ΔGfu and keq
A
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
25
Thermodynamic Stability of Proteins
| Free Energy Equation
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
26
Thermodynamic Stability of Proteins
| Modified Gibbs-Helmholtz Equation
ΔGfu(T) = ΔHfu(1+T/Tm) + ΔCp(Tm-T+Tln(T/Tm))
-where Tm is the melting temperature
27
Heat Adapted Organisms
- thermophiles: 55-80'C
| - hyperthermophiles: 80-113'C
28
Strategies for Increasing Protein Stability
- shifting the free energy curve to higher temperatures
- broadening the free energy curve
- shifting ΔGfu up at all temperatures
29
Thermostabilisation Strategies
- 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)
30
Thermostabilisation Strategies
| Conformational Flexibility and Loop Regions
- 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
31
Themostabilisation Strategies
| Increased Hydrophobicity
- 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
32
Thermostabilisation Strategies
| Ionic Interactions and Networks
- 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
