Protein stability in extreme environments Flashcards
Principles of proteins stability
- Protein stability in general
- Amino acid side chains
- Hydrogen bonds
- Salt bridges
- Hydrophobic interactions
- Sequence differences between mesophiles and extremophiles
- Accessible surface area
- Analysis of structures
- Genome wide studies
Tm : definition
Tm is defined as the temperature at which the free energy of the folded and unfolded states is equal, and half the molecules are folded and half unfolded
Measure using Fluorescence, NMR
Definitions: T1/2
T1/2 is the time taken to lose half activity at a certain temperature
How much free energy is required denature proteins
0.4 kJmol-1 of amino acid – hence 40 kJmol-1 for 100 amino acid protein to change every amino acid in its structure
BUT, H-bond ~12kJmol-1 and many in protein structure + other non-covalent bonds
What happens to fluorescence as temperature increases
- Native protein (dye not bound)
- Partially unfolds and dye binds and flouresces
- Fully denatured protein (maximum dye binding)
- Protein aggregates leading to dye dissociation
How big is the difference in stability between mesophilic proteins and thermostable proteins and what did they use to compare
Small
Compared ferredoxin proteins from Clostridium species with different thermostabilities
What are the observed differences between mesophilic, thermophilic and hypthermophilic protein structures
- Increase in ion pairs
- Reduction in loop size
- Reduction in the number of cavities
- Reduction in the surface area/ volume ratio
- Increased hydrophobic interaction at subunit interfaces
- Increased secondary structure formation
- Truncated N and C termini
- Increase in proline content - some are very long, making them very unstable.
Glutamate dehydrogenase comparisons
Looked at GluDH from Pyrococcus furiosus (hyperthermophile) and GluDH from Clostridium symbiosum (mesophile)
P. furiosus:
Optimum growth temperature 100°C
GluDH T1/2 (100°C) = 12 hours
C. symbiosum
Optimum growth temperature ~37°C
GluDH T1/2 (50°C) = 30 mins
What were the sequence differences between glutamate dehydrogenases
There were more similarities in the top regions than in the bottom region
What was the comparison of ion pair interactions
There were differences in ionic bonds
What do ion pair interactions (salt bridges) require
Interaction between a positively charged residue (Arg/Lys or His) with a negatively charged residue (Glu/Asp) involves both ionic interactions and H bonds
What is a salt bridge
Ionic interaction and hydrogen bonds (not only atoms but also hydrogens that are attached are also involved)
What are the salt bridge networks in P. furiosus GluDH
18 residue salt bridges in the core of the hexamer
3 residue networks, the larger the network, the more stable it becomes (to do with entralpy and enthalpy).
In the network, the entropy is decreased compared to the enthalpy to the pairs so therefore is more stable
What the major difference in GluDH structures between hyperthermophilic and mesophilic
Salt bridge networks
Comparing GluDH between two species with closely related thermostabilities
Compared between Thermococcus litoralis and pyrococcus furiosus
What were the sequence alignment differences between the glutamate dehydrogenases
Amino acids are similar
There were isosteric changes (change is to do with electronegativity)
There were packing changes (the way they pack against each other, these changes lead to alterations)
The change that occurs over and over are the ILE to Val changes - reduced packing efficiency - very similar but the only difference is an extra carbon (makes it bigger). This changes the enthalpy and can adopt different conformations
What are the isosteric changes
Glutamate - negatively charged
Glutamine - positively charged
But they both have the same shape
Cysteine has an O so negatively charged
Serine has a sulfur so can interact with other sulphurs
Valine (non polar) and threonine has oxygen so negatively charged)
What are some complementary sequence changes
They pack against each other (all hydrophobic)
Isosteric changes- valine and leucine (one more carbon atom in the main chain)
Leucine was substituted to methionine (longer than leucine). Isoleucine substituted by a valine, overall nothing has changed.
What are the sequence changes resulting in main chain movements
Main chain movements- isoleucine interacting with the main chain on one side- the pyrococcus pushes away the main chain its packing against
Isoleucine changes to valine (allows the main chain the pack against it more closely)- small changes affect the thermostability
Isoleucine - packing against tyrosine - stacks against the ring. Valine in the thermococcus structure doesn’t change the position of the main chain (so it doesn’t allow water to move through)
Comparion of the ion pair inteactions between the similar thermostable GluDH
Thermococus ion pairs: 38
Pyrcoccus: 45
Ion pairs give extra stability
% of charged charged residues is higher in the pf than the tl
Large networks found in pyrococcus- 5, 6, and 18 residue networks (all multiples of 3-6 subunits)
Tf only has a 16 residue network- similar in shape but has none of the 6 and none of the 5 residue networks
Salt bridge networks in GluDH
6- residue networks are only present in P. furiosus GluDH
What are the salt bridge networks in GluDH
6-residue network was rebuilt in T. litoralis GluDH
T138E mutant is LESS stable, despite network present
It made it worse and didn’t increase the thermostability. Similar ionic properties have oxygens at the end
6- residue network was rebuilt in T. litoralis GluDH Double T138E/D167T mutant results in increased stability
Single changes can make things worse but multiple changes can make things better
Why are salt bridges important at high temperature?
Water molecules form cages around non-polar hydrophobic parts of a molecule
On burying the hydrophobic side chains, caged water molecules are released, increasing the entropy of the solvent
The hydrophobic effect decreases at high temperatures. The ionic interactions in water increase in energy at high temperatures
Whats the dielectric constant of a solvent
The measure of its ability to keep opposite charges apart
Vacuum -1
Water - 80
Non-polar solvent - 4
How do psychrophiles adapt their proteins to the cold
Proteins are generally stable at low temperature
* Enzymes need to be active at low temperature
* Enzymes need to be more flexible and thus less rigid
What are psychrophile defined as having
a) an optimal growth temperature of 15 ° C or lower,
b) a maximal growth temperature below 20 ° C and
c) a minimum growth temperature of 0 ° C or lower.
He defined microbes which grow at 0 - 5 °C, but which
have maximal growth temperatures exceeding 25 ° C as
psychrotrophs (psychrotolerant)
What do psychrophilic enzymes have
Heat labile active sites
Optimisation of activity of psychrophilic enzymes by decreasing substrate affinity
Psychrophilic enzymes have decreased affinity for substrate
Kcat is higher for the psychrophile (more active enzymes). The turnover which they process their substrate is faster.
Km is lower. The Km represents the substrate concentration
Vmax is the maximum rate of reaction - affinity of active site is lower- less affinity, doesnt bind to the enzyme as well
Citrate synthase comparisons
Structures known from many species that span the temperature spectrum
Comparison between DS2-3R and P. furiosus enzymes
How do Psychrophilic adaptations Increase in flexibility
Fewer ion pair networks and inter-subunit ion pairs than hyperthermophile
* More open active site
* More prolines in centre of alpha helices
* Fewer prolines in loops
* BUT – hyperthermophilic enzyme has more glycine
Protection against cold denaturation
* entropic benefit of burying hydrophobic residues is less at low temperature
(lower mobility of liberated water molecules at low T)
* Possibly compensated for by more intra-subunit ion pairs in psychrophile than
mesophile
Role of different types of amino acid residue in thermostability
Proline isomerisation
* Cis-trans isomerization intrinsically slow
* Limiting step in protein folding
* Fewer proline residues in proteins from psychrophiles
* Psychrophiles overexpress prolyl isomerases
What two formations can prolines adopt
Trans or cis
prolines are good if you want sharp bends in the structure
Role of different types of amino acid residue in thermostability
Arginine
* Ion pair – possibility of 5 H bond donors – good for networks
Glutamate /aspartate
* Ion pair – up to 4 H-bond acceptors – good for networks Isoleucine
* Different conformations – can pack in more ways than leucine Good for packing efficiency - avoiding cavities
Role of different types of amino acid residue in thermostability
Glycine
* Side chain (H) more conformational flexibility
Proline
* Side chain bonds to main chain
* less conformational flexibility rigidifying residue in loops
* no main chain N-H hydrogen bond - destabilises helices
Lysine
* Positive charge
* ion pair networks
* long hydrophobic side chain before terminal amine
Why is the context of sequence change important
Most sequence changes involve incremental increase
or decrease in volume of side chain
* limited number of isosteric changes - all involve
charge differences
* Individual sequence changes are most likely destabilizing
* Complementary changes must occur to rescue stability
and allow for evolution towards optimal configuration for
environmental niche
* Structures seen today are result of many eons of evolution
What is Glucose Dehydrogenase from Haloferax mediterranei used for
This enzyme is not that interesting - only because its been used as a model enzyme.
Its a dimeric enzyme of 39 KDa subunit Mr which catalyses the first step in the non- phosphorylated Entner-Doudoroff pathway:
- Glucose + NAD(P)+ D-glucono-1,5-lactone + NAD(P)H + H+
Its a member of the zinc-dependent medium-chain alcohol
dehydrogenase superfamily (MDR family)- takes hydrogen away for its reaction and it needs a zinc. Active site requires a zinc ion in order to carry out its catalytic reaction
H. mediterranei Glucose Dehydrogenase Structure Determination
They did structural studies - they wanted to know what the structure of the protein was like
The protein was crystalised in very high salt conditions inside the bacterium (instead of having a high sodium chloride concentration there is high potassium chloride concentration)
They obtained a high resolution - 1.6A (good view of the molecule and confidence of where the atoms go)
The structures obtained of the free enzyme and a wide range of substrate complexes
It was found to be a dimer- each subunit had 2 domains
Zn and cofactor lie in the cleft between domains
GlcDH structure
2 domains in the subunit
NADP+ Zn and glucose bind deep in the cleft separating the 2 domains
Determining the molecular basis of stability in enzymes
Shows mostly negatively charged residues (aspartic acid) compared to other proteins, there are fewer positively charged residues but there are many negatively charged residues
What structure was GlcDH structurally similar to
H. mediterranei GlcDH is structurally similar to Sulfolobus solfataricus GlcDH and Thermoplasma acidophilium GlcDH
At the time there were very few proteins we could use to model this- the only similar protein at the time was the GluDH in s. solfataricus: not a halophile (lower thermophile, likes to be around 70 maximum)- very similar in structure
Another is in the T. acidophilum: like very low pH - happiest at a pH of 2. The structures very similar to the halophilux - difference is these proteins are not halophilic
Characteristics of the GlcDH solvent accessible surface
Compared to the other proteins, the protein from halophilux has very few lysines
Halophilic adaptation of GlucDH
Surface change
68: extremely negative, most proteins are neutral
-6: this one makes a tetramer- overall each monomer is similar- many more acidic negatively charged residues than in the sulfur- has fewer lysines in the halophilux
Characteristics of the GlcDH solvent structure
- net charge of -70
- only 5 K+ ions found on surface - and only one
uses carboxyl as ligand - 2 of these K+ ions are involved in nucleotide
binding
Has all of these negatively charged residues on the outside - going to interact with the potassium - positively charged (not what they saw in the crystal structures)
Two of them are involved in binding the NADP cofactor- many water molecules in between - structurally important because they’re always in the same position
Halophilic adaptation in Hm GlcDH
- extensive water structure = 1.9 solvent per residue (average of
all structures at similar resolution = 1.2) - water structure is very well ordered - low B factors
- highest relative proportion of solvent in second shell
Adaptation to negative surface. No counter ions, extensive ordered water structure
Solvation or Hydradion Shells of Proteins
Water molecules are in shells- oxygen part tends to be negatively charged whereas the hydrogen parts tend to be slightly positively charged
Water molecules arrange themselves around these in the opposite way
In the sodium - opposite- make the hydrogen bonds.
A protein with a mostly negatively charged surface will have all of these in the first shell reacting directly with the negative residues- all the hydrogens pointing at the residues (have a second layer around them where the water molecules are interacting with the first layer of water molecules)- can have up to 3/ 4 of these layers
Well constructed solvent layer/ shell around them- this is what allows the proteins to work in the way they were working earlier - little water in large amount of solvent
Characteristics of the GlcDH solvent accessible surface
Surface Properties
* Increase in negative surface
* Decrease in hydrophobic surface from decrease in
surface lysine residues
Same characteristics also observed in the D-2hydroxyacid
dehydrogenase from Haloferax medittereanei
D 2-hydroxyacid dehydrogenase from H. mediterranei Structure Determination
They crystallised it - wasn’t any need for a high potassium chloride concentration but there was a high amount of the magnesium acetate
D2HDH has two domains - the active site lies between these and managed to obtain a resolution of 1.2 armstrongs
Characteristics of the GlcDH solvent accessible surface
Similarities between two halophilc proteins
Negatively charged residues are increased as a opposed to the non halophilic
Reduction in lysines than in the non halophilic
Archaeal Halophilic adaptation – water structure
Adaptation to negative surface
No counter ions
Extensive ordered water
structure
Neutron Diffraction Experiments
show that hydration around carboxylate group of ASP is enhanced at high salt.
Genome wide amino acid prevalence in halophiles against mesophilic orthologs
Based on the DNA of all the different halophiles, can guess which ones are going to be proteins and decipher which amino acids they’re going to have
There’s a drastic reduction in halophilic reactions and increase in the negatively charged residues
