Protein stability in extreme environments

Question 1

Principles of proteins stability

Answer 1

1. Protein stability in general
2. Amino acid side chains
3. Hydrogen bonds
4. Salt bridges
5. Hydrophobic interactions
6. Sequence differences between mesophiles and extremophiles
7. Accessible surface area
8. Analysis of structures
9. Genome wide studies

Question 2

Tm : definition

Answer 2

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

Question 3

Definitions: T1/2

Answer 3

T1/2 is the time taken to lose half activity at a certain temperature

Question 4

How much free energy is required denature proteins

Answer 4

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

Question 5

What happens to fluorescence as temperature increases

Answer 5

1. Native protein (dye not bound)
2. Partially unfolds and dye binds and flouresces
3. Fully denatured protein (maximum dye binding)
4. Protein aggregates leading to dye dissociation

Question 6

How big is the difference in stability between mesophilic proteins and thermostable proteins and what did they use to compare

Answer 6

Small

Compared ferredoxin proteins from Clostridium species with different thermostabilities

Question 7

What are the observed differences between mesophilic, thermophilic and hypthermophilic protein structures

Answer 7

1. Increase in ion pairs
2. Reduction in loop size
3. Reduction in the number of cavities
4. Reduction in the surface area/ volume ratio
5. Increased hydrophobic interaction at subunit interfaces
6. Increased secondary structure formation
7. Truncated N and C termini
8. Increase in proline content - some are very long, making them very unstable.

Question 8

Glutamate dehydrogenase comparisons

Answer 8

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

Question 9

What were the sequence differences between glutamate dehydrogenases

Answer 9

There were more similarities in the top regions than in the bottom region

Question 10

What was the comparison of ion pair interactions

Answer 10

There were differences in ionic bonds

Question 11

What do ion pair interactions (salt bridges) require

Answer 11

Interaction between a positively charged residue (Arg/Lys or His) with a negatively charged residue (Glu/Asp) involves both ionic interactions and H bonds

Question 12

What is a salt bridge

Answer 12

Ionic interaction and hydrogen bonds (not only atoms but also hydrogens that are attached are also involved)

Question 13

What are the salt bridge networks in P. furiosus GluDH

Answer 13

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

Question 14

What the major difference in GluDH structures between hyperthermophilic and mesophilic

Answer 14

Salt bridge networks

Question 15

Comparing GluDH between two species with closely related thermostabilities

Answer 15

Compared between Thermococcus litoralis and pyrococcus furiosus

Question 16

What were the sequence alignment differences between the glutamate dehydrogenases

Answer 16

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

Question 17

What are the isosteric changes

Answer 17

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)

Question 18

What are some complementary sequence changes

Answer 18

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.

Question 19

What are the sequence changes resulting in main chain movements

Answer 19

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)

Question 20

Comparion of the ion pair inteactions between the similar thermostable GluDH

Answer 20

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

Question 21

Salt bridge networks in GluDH

Answer 21

6- residue networks are only present in P. furiosus GluDH

Question 22

What are the salt bridge networks in GluDH

Answer 22

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

Question 23

Why are salt bridges important at high temperature?

Answer 23

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

Question 24

Whats the dielectric constant of a solvent

Answer 24

The measure of its ability to keep opposite charges apart

Vacuum -1
Water - 80
Non-polar solvent - 4

Question 25

How do psychrophiles adapt their proteins to the cold

Answer 25

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

Question 26

What are psychrophile defined as having

Answer 26

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)

Question 27

What do psychrophilic enzymes have

Answer 27

Heat labile active sites

Question 28

Optimisation of activity of psychrophilic enzymes by decreasing substrate affinity

Answer 28

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

Question 29

Citrate synthase comparisons

Answer 29

Structures known from many species that span the temperature spectrum
Comparison between DS2-3R and P. furiosus enzymes

Question 30

How do Psychrophilic adaptations Increase in flexibility

Answer 30

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

Question 31

Role of different types of amino acid residue in thermostability

Answer 31

Proline isomerisation
* Cis-trans isomerization intrinsically slow
* Limiting step in protein folding
* Fewer proline residues in proteins from psychrophiles
* Psychrophiles overexpress prolyl isomerases

Question 32

What two formations can prolines adopt

Answer 32

Trans or cis

prolines are good if you want sharp bends in the structure

Question 33

Role of different types of amino acid residue in thermostability

Answer 33

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

Question 34

Role of different types of amino acid residue in thermostability

Answer 34

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

Question 35

Why is the context of sequence change important

Answer 35

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

Question 36

What is Glucose Dehydrogenase from Haloferax mediterranei used for

Answer 36

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

Question 37

H. mediterranei Glucose Dehydrogenase Structure Determination

Answer 37

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

Question 38

GlcDH structure

Answer 38

2 domains in the subunit

NADP+ Zn and glucose bind deep in the cleft separating the 2 domains

Question 39

Determining the molecular basis of stability in enzymes

Answer 39

Shows mostly negatively charged residues (aspartic acid) compared to other proteins, there are fewer positively charged residues but there are many negatively charged residues

Question 40

What structure was GlcDH structurally similar to

Answer 40

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

Question 41

Characteristics of the GlcDH solvent accessible surface

Answer 41

Compared to the other proteins, the protein from halophilux has very few lysines

Question 42

Halophilic adaptation of GlucDH

Answer 42

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

Question 43

Characteristics of the GlcDH solvent structure

Answer 43

• 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

Question 44

Halophilic adaptation in Hm GlcDH

Answer 44

• 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

Question 45

Solvation or Hydradion Shells of Proteins

Answer 45

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

Question 46

Characteristics of the GlcDH solvent accessible surface

Answer 46

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

Question 47

D 2-hydroxyacid dehydrogenase from H. mediterranei Structure Determination

Answer 47

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

Question 48

Characteristics of the GlcDH solvent accessible surface

Answer 48

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

Question 49

Archaeal Halophilic adaptation – water structure

Answer 49

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.

Question 50

Genome wide amino acid prevalence in halophiles against mesophilic orthologs

Answer 50

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