Proteins: Terry & Paul G v.1 Flashcards
Define: Gibbs free energy (ΔG)
Free energy change for a reaction:
ΔG = Gproducts - Greactants
What is the equation to find the activation energy for a reaction?
ΔG‡ = Gtransition state - Greactant
Gibbs Free Energy, Enthalpy and Entropy equation
ΔG = ΔH - TΔS
ΔG: change in Gibbs free energy
ΔH: change in enthalpy (heat)
T: absolute temperature (K)
ΔS: change in entropy (disorder)
How do spontaneous reactions occur?
Negative ΔG, which can be given by:
- Big negative ΔH (exothermic)
- OR Big positive ΔS (increase in entropy)
What is the first law of thermodynamics?
Law of Conservation of Energy, states that energy can neither be created nor destroyed; energy can only be transferred or changed from one form to another.
ΔE=q+w
- ΔE: change in internal energy
- q: heat
- w: work
What is the second law of thermodynamics?
The entropy of an isolated system always increases
What is the third law of thermodynamics?
The entropy of a system approaches a constant value as the temperature approaches absolute zero.
Define: exergonic
- ΔG is negative
- Free energy released
- Favourable: Spontaneous
Define: endergonic
- ΔG is positive
- Free energy absorbed
- Unfavourable: not spontaneous
Define: exothermic
- ΔH is negative
- Heat absorbed
Define: endothermic
- ΔH is positive
- Heat absorbed
How do you calculate the thermodynamic equilibrium constant from aA + bB ⇌ cC + dD?
[X]eq=concentration of substrate X at equilibrium
Calculating ΔG from ΔG°:
ΔG = ΔG° + RT ln Qi
Qi: where Q is calculated with initial concentrations of the reactants and products
Relating free energy with the equilibrium constant:
ΔG° = -RT ln K
What are the CHEMISTRY and PHYSICS standard conditions? ΔG°
- 298K
- Gases at partial pressure of 101.3 kPa (1 atm)
- Reactants & products at 1M
- [H+] = 1 M ⇒ pH=0
What are the BIOCHEMISTRY standard conditions? ΔG’°
- 298K
- Gases at partial pressure of 101.3 kPa (1 atm)
- Reactions occur at well-buffered aqueous solution at pH 7 e.g. [H+] & Mg2+
- [H+] = 10-7
- Mg2+ = 1 mM
Why are we interested in free energy?
It has predictive power. If we know standard free energy, we know:
- Under what initial conditions can the reaction occur spontaneously?
- Does reaction require coupling with a favoured reaction?
- What is the position of reaction at equilibrium?
- Theoretically, how much work can it do?
Making “unfavourable” reactions go: relating to Q/K
*the measure of whether a reaction will proceed spontaneously is ΔG, not ΔG’°
- If ΔG’° is positive, ΔG can be negative by altering initial conditions.
- For RT ln Q to be negative: Q < 1, therefore ln Q would be negative
- The concentration of products must be kept much lower than reactants:
- Removing products faster than it’s produced
- Replenishing/adding reactants faster than it’s being used up
Making “unfavourable” reactions go: coupling
- (Think story about Terry and his wife)
- Couple unfavourable with highly favourable reaction (in the active site of an enzyme)
- Must have a shared components (reactions and products)
Give 3 examples of how ‘unfavourable’ reactions are made ‘favourable’
- Reaction couplings
- Constant replenishing of reactants at a faster rate than they are being used
- Constant removal of products at a faster rate than they are being produced
Define: nucleotide
base + sugar + phosphate
Define: nucleoside
base + sugar
Explain why ATP is an energy-rich molecule
- Energy is released upon hydrolysis of phosphoanhydride bonds (uses energy)
- breakdown of ATP to:
- ADP + Pi (inorganic phosphate)
- AMP + PPi (pyrophosphate)
- breakdown of ATP to:
- Molecules at the start are less stable than the molecules that are formed
- Strong bonds are formed: creates a lot of energy
- Less negative charge (and repulsion) in ADP (or AMP) than ATP ⇒ more stable
- Pi ⇒ bonds in the inorganic phosphate are a lot stronger than the phosphoanhydride bonds (resonance forms)
- Substantially more energy is created from the formation of strong bonds than the breaking of weak bonds
Breaking a bond…
A. Releases energy
B. Absorbs energy
B
Bonds are happy places for atoms, as in it’s more stable, therefore energy must be put into the system to break bonds
Forming a bond…
A. Releases energy
B. Absorbs energy
A
Forming a bond releases energy
Why does the hydrolysis of ATP yield energy?
- ATP phosphoanhydride bonds are relatively weak and strong bonds are formed (net energy released)
- ATP has a higher negative charge density than ADP (ATP less stable)
- Pi (inorganic phosphate) is very stable; multiple resonance states exist
- The reaction is highly exergonic
- Products are much more stable than reactants
Define: hydrolysis
The addition of H2O into a molecule
Define: erythrocytes
blood cells
Does a positive charge or a negative charge in free energy indicate a favourable (spontaneous) chemical event?
A negative change in free energy (ΔG) indicates a favourable (spontaneous) chemical event
How is the free energy of the reverse reaction related to that of the forward reaction?
Sign changes from + to - and vice versa
If some spontaneous event is endothermic, what must have been the “driving force” behind that event, enthalpy or entropy?
Highly positive entropy value (ΔS), because endothermic reactions have ΔH > 0 and only exothermic (ΔH < 0) values contribute to spontaneous reactions
Given the free energy changes for coupled reactions that result in the conversion of glucose to glucose 6-phosphate:
- Is the K’eq for reaction (1) bigger or smaller than K’eq for equation (2)?
- Which reaction has the largest K’eq, reaction (1), (2) or (3)?
- Which reaction has the smallest K’eq reaction (1), (2) or (3)?
- K’eq for reaction (1) is smaller than the K’eq for reaction (2)
- reaction (2)
- reaction (1)
Define: the basic unit for DNA and RNA
nucleic acid (bases)
Define: The basic unit for proteins
amino acid (residues)
How can protein be post-translationally modified?
- Phosphorylation (signalling)
- Glycosylation (extracellular protection, signalling) - change of nature
- Proteolytically cleaved (trafficking, inhibition) - decides the location of a protein in the body
- Acylation (fatty acids, localisation e.g. to a membrane, regulation) - turns on/off proteins, and also the location of where in the body the protein goes
Define: proteomics
Analysis of the complete complement of expressed proteins and their interactions
Protein shape/structure: is it rigid or flexible?
- flexible
- can change conformations
- considered as soft matter
- the motion of a protein is based on its function (flexibility/dynamics is on a timescale of function)
- but still HIGHLY STRUCTURED
Define: somatic cells
All cells in an organism, except the sex cells
All somatic cells types (except germs) from one organism:
- Might not have the same DNA and protein content
- Have the same DNA content, but not necessarily the same protein content
- Might not have the same DNA, but have the same protein content
- Have the same DNA and protein content
B
All somatic cell types (not germ cells) from an organism have the same DNA, but not necessarily the same protein content
Proteins can be post-translationally modified, which can change their… (3 things)
- chemical properties
- conformation
- function
Define: N-terminal
amino-terminal of a protein
Define: C-terminus
the carboxyl terminus of a protein
Define: primaryprotein structure
the amino acid sequence in order from N-terminus to C-terminus
Define: secondary protein structure
local areas of regular ordered structure
Define: tertiary protein structure
The 3D fold of a protein subunit
Define: quaternary protein structure
The organisation of subunits (controls protein function)
Amino acids are chiral. TRUE or FALSE?
TRUE
(except for glycine)
Define: L-configurations (in relation to amino acids)
Define: D-configurations (in terms of amino acids)
All proteins (or an overwhelmingly large majority) are in:
- D-configuration
- L-configuration
B
D-configurations usually appear in toxins and the like
What is the number of common amino acids?
20
What does it mean to say that amino acids are zwitterionic?
both the carboxylate and amine ends of an amino acid can be ionized, which means it is neutral and has no net charge at neutral (≈7) pH
Define: amino acid residue
amino acids in the context of peptides and proteins
What is the Henderson-Hasslebalch equation?
pKa = pH, when [A-] = [HA]
Off all the following molecules, which one can’t exist:
- +H3N—CHR—COOH
- +H3N—CHR—COO-
- H2N—CHR—COO-
- H2N—CHR—COOH
D
NH2—(CHR)—COOH
Define: glycosylation
Addition of a carbohydrate (enzymatic addition of group) to a serine, threonine or asparagine residue
Define: phosphorylation
the addition of a phosphate group (involving enzymatic activity) to a serine, threonine or tyrosine residue
What is the mean molecular weight of an amino acid residue? Do we use this value to make approximations on the molecular weights of proteins?
The mean MW of an amino acid residue is 118 Da, however, we use 110 Da per residue to calculate molecular weight approximations for proteins as this value takes into account the different frequencies of the amino acids
Define: condensation reactions
a chemical reaction where 2 molecules are joined together by a covalent bond to make a larger, more complex, molecule, with the loss of a small molecule
Define: peptide bond
a bond created through a condensation reaction between two amino acids, occurring with the loss of a water molecule
Where is the peptide bond formed naturally?
The peptide bond is naturally made in the ribosomes (during translation), it can however, be synthesised in the lab
What kind of bond is a peptide bond?
- hydrogen bond
- phosphodiester bond
- covalent bond
- polar bond
C
a peptide bond is a stable covalent bond
Although peptide bonds are covalent they can be broken up. How?
They are hydrolysed by enzymes called proteases
What causes peptide bonds to have partial double bond character? What does it affect?
The TWO resonance structures cause peptide bonds to have a partial double bond character. This affects the length of the peptide bond (1.32Å, instead of 1.49Å for a SB and 1.27Å for a DB), and causes RIGIDITY and PLANARITY in the structure
Which one of the following is the correct peptide backbone sequence?
- Cα - C - Cα - C - Cα - C
- N - Cα - C - N - Cα - C
- C - C - Cα - C - C - Cα
- N - C - Cα - N - C - Cα
B
N - Cα - C - N - Cα - C
How many ways are peptide bonds oriented and what are they called? Which one is the most common?
Two ways:
- cis ⇒ ω = 0° (α-carbons are on the same side of the peptide bond)
- trans ⇒ ω = 180° (α-carbons are on opposite sides of the peptide bond)
The TRANS configuration is the most common transfiguration
Why is the trans peptide bond configuration more preferable?
it is sterically or energetically favoured because the groups are far from each other (minimised crowding)
What is interesting about the peptide bond that precedes the proline amino acid?
Both the cis and trans peptide bond configuration has a steric clash, making trans configurations in prolines not as stable as normal trans configurations
What does it mean when we say that the primary amino acid sequence is asymmetric?
It means that the sequence from the N-terminus to the C-terminus is different from the sequence that runs from the C-terminus to the N-terminus
The amino acid sequence is written:
- always from the amino-terminal end to the carboxyl end.
- always from the carboxyl-terminal end to the amino-terminal end.
- in both direction.
A
It is always written from the N-terminus to the C-terminus
What bonds are part of the covalent structure of proteins?
- peptide bonds
- hydrogen bonds
- disulphide bridges
- hydrophobic bonds
A & C
peptide bonds and disulphide bridges (between 2 cysteines - not necessarily adjacent in the primary sequence)
What can impact the molecular weight calculations of proteins?
- prosthetic groups (e.g. heme group = 620 Da, metallic groups)
- post-translational modifications
What happens to the pKa of an amino acid/peptide as it lengthens? Why does this occur?
The pKa of the carboxylic acid end will increase and the pKa of the amino end will decrease, causing a narrower region where the amino acid is ionised. This happens because the charges at the two terminal ends are further away and cannot stabilise each other as well.
NOTE: Opposite charges can stabilise each other (therefore lone charges are poorly tolerated in proteins and want to be neutral)
Define: isoelectric point (pI)
the pH where the protein carries NO net charge
Acidic protein pIs are:
- low
- high
A
Basic protein pIs are:
- low
- high
B
What happens to the charge the protein carries when the pH is above and when the pH is below the pI?
- At pH below the pI, the protein carries a net positive charge
- At pH above the pI, the protein carries a net negative charge
Why is the pI of a protein so important?
It is functionally important for where a protein lives and its behaviour
e.g. pepsin lives in the stomach (which is a very acidic environment)
The theoretical pI for human pepsin is 3. What makes the pI of this protein so low?
It is rich in acidic amino acids (e.g. Asp (D) and Glu (E))
The theoretical pI for chicken lysozyme is 10. What makes the pI of this protein so high?
The protein is rich in basic amino acid residues, like Arg (R) and Lys (K)
What does the symbol “Φ” (phi) signify in the protein structure?
the rotation around the bond between N and Cα
What does the symbol “Ψ” (psi) signify in the protein structure?
the rotation around the bond between Cα and C
Define: torsion angle
A dihedral (torsion angle) is made up of four atoms or three bonds
What are the properties of the torsion angle ω (omega)?
- It is the torsion angle in the peptide bond (between N and C)
- There is NO rotation around the peptide bond (it is rigid)
- It has two orientations, cis (0°) and trans (180°) - the peptide bond conformations
Is there free rotation around the φ-ψ torsion angles?
No. This is due partially to the rigidity of the ω torsion angle and also because of the steric clashes between the atoms (especially the R-groups)
Draw up a Ramachandran plot/Φ-Ψ plot
- include the axis labels
- add in the α, β and L (left-handed helix) regions
- explain why there are sometimes amino acids shown in the “forbidden”/”disallowed” region
Glycine will show up in the forbidden region because it has no real R-group (less steric clash)
What kinds of structures can be found in the blank spaces in this Ramachandran plot?
Nothing! It’s forbidden, although a few structures may rarely appear in the RH side of the plot, none will appear at ΦΨ zero degree angles.
Define: regular secondary structure
repeating φ,ψ angles for sequential residues
Not a repeating pattern of φ,ψ angles but the same repeatedly
Define: irregular secondary structure
non-repeating Φ, Ψ angles for sequential residues
The irregular structure still has Φ, Ψ angles in the allowed regions, but sequential residues have markedly different angles
What are the properties of α-helices?
- right-handed helix
- Φ, Ψ angle pairs are around -60°, -50°
- all NH and C=O within the helix (except those at the end) form favourable internal hydrogen bonds
- hydrogen bond groups are parallel to the helical axis
- also called the 3.613 helix
- 3.6 residues in a turn and 13 atoms in a hydrogen bond “ring”
- amphipathic
Define: amphipathic
has a hydrophilic and a hydrophobic side
How can we guess if an α-helix is present in a protein from its primary sequence?
by the repetition of a hydrophobic residue every three to four residues
What are the features of β-sheets?
- A β-sheet consists of two or more β-strands. The strand is the element.
- Strands are parallel/antiparallel to neighbouring strands. Sheets can be pure/mixed. - twisted sheets are abundant (usually a RH twist)
- φ,ψ -130º, +130º (approx.)
- Hydrogen bonds between C=O and NH of opposing strands: antiparallel more stable; outer NH and C=O of a sheet are not hydrogen bonded.
- side-chains are pointed up and down with respect to the sheet
What are the features of β-turns?
- helps reverse the mainchain - redirects backbone
- abundant in proteins (mostly on the surface - particularly for β-sheets)
- consists of four residues
- residues i+1 and i+2 have different Φ, Ψ angles
- a hydrogen bond between the carbonyl of the first (i) and the NH of the fourth (i+3) stabilise the turn
- Proline is often in position i+1
What is the difference between type I and type II β-turns?
- type II β-turns have a steric clash between the i+1 and i+2 residues and often have Gly in position i+2
What interactions (non-covalent) drives proteins to fold into their tertiary (native) structure?
- van der Waal contacts - how close can atoms pack
- hydrogen bonds - allow atoms to pack within VDW distances
- salt bridges - stabilisation of charges
- hydrophobic effect - the main driving force for stabilising the folded protein
Define: Van der Waals interactions
- weak, short-range electrostatic attractive forces between uncharged molecules, arising from the interaction of permanent or transient electric dipole moments
- two atoms can approach each other to within a distance called the Van der Waals radii
- up to that distance there is a favourable attraction
Define: Rm (contact distance)
Rm (contact distance) is the minimum energy required to sustain an attraction
Draw the basic graph shape of a typical Van der Waals interaction. Mark where Rm is and at which point the interactions are both favourable and unfavourable.
Note: Do not need to include values
Define: hydrogen bonds
A hydrogen bond occurs when two electronegative atoms compete for the same hydrogen atom
What is the most favourable/common geometry for a hydrogen bond?
collinear
Define: electrostatic interactions
- AKA “salt bridges” or “ion pairs”
- Ionic interactions between oppositely charged groups in proteins
- stabilises the ionic interaction between two oppositely charged species (charges are distributed over several atoms - which is more favourable)
Why do proteins fold?
- proteins contain nonpolar groups
- water is a poor solvent for nonpolar groups and nonpolar groups prefer to interact with nonpolar groups
- the process of protein folding is driven by entropy (more specifically, solvent entropy)
The amino acid sequence is important in determining the 3D structure of a protein. TRUE or FALSE?
TRUE
Why is there an entropic requirement to bury hydrophobic groups?
Because, when hydrophobic groups aren’t buried, more water molecules will be ordered around the protein, burying hydrophobic groups releases many “ordered” water molecules, which increases the level of disorder in the solvent
All buried polar groups form hydrogen bonds. TRUE or FALSE?
TRUE
Proteins are very stable molecules. TRUE or FALSE?
FALSE
They are marginally stable and are in an equilibrium with it’s unfolded state, i.e. there will most likely be an unfolded protein in a sample of proteins. However, the folded native state is much preferred.
What methods have been used to determine protein structures?
- X-Ray Crystallography
- NMR Spectroscopy
- CryoElectron Microscopy (CryoEM)
What are 3 ways to represent protein structure?
- Wire/Line/Stick: full details of atom positions
- Cartoon/Ribbon: content, positions and orientations of helices and strands
- Sphere: nature of the surface, residue exposed, coloured in the type of residues (e.g. hydrophobic, hydrophilic etc.)
What are the key features of myoglobin?
- oxygen-binding protein of muscle cells
- eight α-helices (70% of the residues are in the helices)
- 154 amino acid (aa) residues
- Has a heme prosthetic group
What are the 3 simple super-secondary structures that make up many proteins?
- βαβ element
- ββ-hairpin
- α-α corner/hairpin
What are the four rules of protein structure?
Note: these are generalised rules and will have exceptions
- hydrophobic interactions contribute to stability: exclusion of water by burial of hydrophobic side chains requires at least two layers of secondary structure
- when α-helices and β-sheets occur together, they are found in separate layers: backbone hydrogen bonding patterns do not allow helices to hydrogen bond to sheets
- segments of the protein that are adjacent in the sequence are usually adjacent in the structure
- individual β-strands favour a right-hand twist - two parallel strands are then generally and preferably right-hand connected ⇒ such connections are shorter than a left-hand connection
Define: globular proteins
- folded into globular or roughly spherical shapes
- function as enzymes and in cellular control
Define: fibrous proteins
- polypeptides in long strands or sheets
- structural proteins with functions of support, defining shape and protection
Define: integral membrane proteins
functions as receptors and transporters
Define: domain
a domain or fold refers to the arrangement and order of secondary structural elements that contain a single hydrophobic core
What processes create proteins with different functional properties (but without new domains being created)?
- intragenic mutations
- gene duplication
- DNA segment shuffle
- gene lateral transfer
What does it mean to say that most proteins are modular?
They have repeating domains (e.g. transcription factors) ⇒ each may have a slightly different sequence, but they all have the same/similar fold
Define: intragenic mutation
point mutations, insertions, deletions
Define: gene duplication
whole or part of a genome is duplicated
Define: DNA segment shuffle
two or more existing genes can be broken and recombined
Think of the broken fragments as domains
Define: gene lateral transfer
one organism acquires parts of the genome of another
Define (in terms of sequence alignment): identity
exactly the same residue (invariant)
Define (in terms of sequence alignment): similar
a change in residue that is observed frequently or with similar physical-chemical properties e.g. Ser to Thr, Val to Leu (conservative)
How are proteins aligned? Why are gaps used in sequence alignment?
- proteins are aligned based on identical/similar residues in the same position
- gaps are used to account for residue insertion and deletions
How similar do proteins have to be for us to determine that they may be related?
They should share greater than 25% of identical residues
What can we determine if we find that two or more protein domain sequences show >25% sequence identity?
- they diverged from a common ancestral domain
- they have a similar fold
Define: homologues
two or more protein domains that share >25% identity
Define: orthologue
homologous proteins that perform the same function in different species
Define: paralogue
homologous proteins that perform different but related functions within one organism
What changes faster?
- protein sequence
- protein structure
A
protein sequence
Residues within a sequence change at the same rate. TRUE or FALSE?
FALSE
Functional residues are highly conserved both in protein sequence and protein structure
Define: quaternary structure
- the assembly of subunits in a protein with two or more polypeptide chains (or subunits)
Define: subunit
a polypeptide chain that is part of a protein with quaternary structure
What does the quaternary structure of hemoglobin consist of?
Hemoglobin is a tetramer of four subunits: two α-subunits and two β-subunits. Hemoglobin can also be described as having two αβ protomers
Define: oligomer
general term for a multimeric protein that consists of a small number of subunits
Define: protomer
a protomer is the structural unit of a protein with quaternary structure
Conformational change of one subunit can be transferred through an oligomer to affect the whole complex. TRUE or FALSE?
TRUE
e.g. Hemoglobin undergoes a conformational change when oxygen binds
Which state of hemoglobin has the highest affinity for oxygen? T or R state?
R state (oxyhemoglobin)
Where is myoglobin expressed?
Myoglobin is expressed only in cardiac and oxidative skeletal muscle
What are the two main functions of myoglobin?
- storage of oxygen in muscles
- release of oxygen when rapidly contracting muscle need energy
Myoglobin is abundant in:
- human muscle cells
- the bloodstream
- the bloodstream of animals living at high altitude
- muscles of diving animals
D
muscles of diving animals
Proteins are usually repellant. TRUE or FALSE?
FALSE
precipitated proteins are bad: they lose their function and are hard to dissolve into a solute environment
Define: ligand
an ion or molecule that attaches to the binding site of a protein
Define: prosthetic group
A prosthetic group is a tightly bound, specific non-polypeptide unit required for the biological function of some proteins. The prosthetic group may be organic (such as a vitamin, sugar, or lipid) or inorganic (such as a metal ion), but is not composed of amino acids.
Define: equilibrium association constant
include units
Units: M-1
Define: equilibrium dissociation constant
Units: M
What does the Kd of a protein-ligand binding represent?
It represents the concentration of free ligand at which the protein is 50% saturated
How do you calculate the fraction of protein binding sites occupied?
Does a smaller or larger Kd represent a higher affinity for ligand binding?
smaller
Explain how cooperative binding differs from standard ligand binding
Cooperative binding graphs show a sigmoidal curve whereas standard ligand binding is shown as a logarithmic curve. Cooperative binding also has some sort of conformation change that can alter the protein’s affinity for a certain ligand.
How do hemoglobins bind and release oxygen (O2)?
- Hemoglobin switches from low-affinity to a high-affinity state as more O2 molecules bind
- The first O2 binds a globin subunit of deoxyhemoglobin in the T state weakly, and makes the T state unstable, so the “T-R” transition of the hemoglobin molecule is made easier
What kinds of major shifts occur as hemoglobin shifts from the T to R state?
- His HC3 residues of the β subunits are involved in ion pairs in the T state
- When oxygen binds, the His HC3 residues rotate towards the centre of hemoglobin and are no longer involved in ion pairs (R state)
- The α1β1 and α2β2 subunit pairs slide over each other and rotate, closing a pocket in the hemoglobin
