the holy grail: quantitative stability/flexibility relationships (qsfr)
DESCRIPTION
The Holy Grail: Quantitative Stability/Flexibility Relationships (QSFR). The mDCM is a heterogeneous mean field theory that directly relates thermal stability to mechanical flexibility. Livesay et al. (2004). FEBS Letters 576:468-476. Jacobs & Dallakyan (2005). Biophysical J. 88:903-915. - PowerPoint PPT PresentationTRANSCRIPT
The Holy Grail:Quantitative Stability/Flexibility Relationships (QSFR)
The mDCM is a heterogeneous mean field theory thatdirectly relates thermal stability to mechanical flexibility
more native -like torsion constraints
more intramolecular H
-bond crosslinking
Nnt
Nhb
native -likeconstraint topologies
denaturedconstraint topologies
Livesay et al. (2004). FEBS Letters 576:468-476.Jacobs & Dallakyan (2005). Biophysical J. 88:903-915.
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Z =Nhb =1
Nhbmax
∑ e− G(Nhb ,Nnt )
RT
Nnt =1
Nntmax
∑
G = −RT lnZ
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G(Nhb,Nnt ) =U(Nhb ) + u(Nhbmax −Nhb ) + v(Nnt )
−T Sconf (Nhb,Nnt | γ,δnat ,δdis) + Smix (Nhb,Nnt ){ }
mDCM work flow…
Livesay et al. (2004). FEBS Letters 576:468-476.Jacobs & Dallakyan (2005). Biophysical J. 88:903-915.
What are the DCM parameters?Fitting ubiquitin and HBP Cp measurements
Jacobs & Dallakyan (2005). Biophysical J. 88:903-915.
Introducing a global flexibility order parameter = #IDF / residue
Rel
ativ
e fr
ee e
nerg
y (k
cal/m
ol)
Comparison of ubiquitin and HBP free energy landscapes
Global flexibility order parameter
1.0 2.0 3.0
Global flexibility order parameter
1.0 2.0 2.51.5
Rel
ativ
e fr
ee e
nerg
y (k
cal/m
ol)
1.0
2.0
0.0
1.0
2.0
0.0
3.0
4.0
5.0
0.5
331 K335 K338 K
330 K
Ubiquitin Histidine binding protein
Jacobs & Dallakyan (2005). Biophysical J. 88:903-915.
Rel
ativ
e fr
ee e
nerg
y (k
cal/m
ol)
Global flexibility order parameter
1.0 2.0 3.0
Global flexibility order parameter
1.0 2.0 2.51.5
Rel
ativ
e fr
ee e
nerg
y (k
cal/m
ol)
1.0
2.0
0.0
1.0
2.0
0.0
3.0
4.0
5.0
0.5
331 K335 K338 K400 K
330 K
Comparison of ubiquitin and HBP free energy landscapes
Ubiquitin Histidine binding protein
Jacobs & Dallakyan (2005). Biophysical J. 88:903-915.
Model generality:Holding max and dis fixed while fitting the remaining 3 parameters
Livesay et al. (2004). FEBS Letters 576:468-476.
Flexibility Index Prob. to Rotate
Prob. of IDOF G(na)
ThioredoxinJacobs, Livesay, et al. (2006). J Mol Biol 358, 882–904
Quantifying MolecularCooperativity
Gly33 Met37
Gln50 Arg73
Jacobs, Livesay, et al. (2006). J Mol Biol 358, 882–904
The extent of cooperativity correlation within the ensemble is dependent on temperature
Introducing QSFR…
The mesophilic/thermophilic RNase H pair
* *1ril -RKRVALFTDGACLGNPGPGGWAALLRFHAHEKLLSGGEACTTNNRMELKAAIEGLKALKE2rn2 MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKE *1ril PCEVDLYTDSHYLKKAFTEGWLEGWRKRGWRTAEGKPVKNRDLWEALLLAMAPHRVRFHFV2rn2 HCEVILSTDSQYVRQGITQ-WIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWV
1ril KGHTGHPENERVDREARRQAQSQAKT--------2rn2 KGHAGHPENERCDELARAAAMNPTLEDTGYQVEV
Alpha-helix; Beta-strand; Catalytic site
Identity conservedChemically conserved
Without conservation
Not all theories are created equal…A simple electrostatics-only model actually predicts the mesophilic
ortholog to be more stable than its thermophilic counterpart!
pH 3.5
pH 3.5
Poisson-Boltzmann ElectrostaticsThe Distance Constraint Model
Parameter differences are believed to retain physical meaning. Because these experiments are done under identical experimental conditions and their structures are remarkably similar, parameter differences should reflect intrinsic thermodynamic differences between the pair.
Livesay, Jacobs (2006). Proteins 62:130-143.
-15
0
15
30
45
0 1 2 3 4 5 6 7 8 9 10
pH
deltaG(elec) (kcal/mol)
Thermophilic (1ril)
Mesophilic (2rn2)
Difference (Thermo - Meso)
0
10
20
30
290 310 330 350 370
Temperature (K)
Heat capacity (kcal/mol*K)
Ishikawa et al., (1993) J. Mol. Biology 230:529-542
“Thus the exquisite rearrangement of the hydrophobic side-chains, including some favorable aromatic-aromatic interactions, is likely to contribute to the increased stability of T. thermophilus RNase H.”
Livesay, Jacobs (2006). Proteins 62:130-143.
Livesay, Jacobs (2006). Proteins 62:130-143.
Hollien & Marqusee (1999), PNAS
The mDCM can identify allostery through flexibility correlation
Livesay, Jacobs (2006). Proteins 62:130-143.
Livesay, Jacobs (2006). Proteins 62:130-143.
The mDCM can identify allostery through flexibility correlation
Parameter error analysis indicates that theflexibility quantities are remarkably robust
PR
(nati
ve b
asi
n o
nly
)P
R (n
ati
ve b
asi
n o
nly
)
Livesay, Jacobs (2006). Proteins 62:130-143.
Parameter error analysis indicates that theflexibility quantities are remarkably robust
Livesay, Jacobs (2006). Proteins 62:130-143.
A random good fit Another random good fit
A bad fit The best fit
10 random good vs. a bad fit
E. coli
T. thermophilus
Parameter error analysis indicates that theflexibility quantities are remarkably robust
Livesay, Jacobs (2006). Proteins 62:130-143.
Supp. Fig. 1. Best fit u,v pairs are plotted against a coarse-grained nat range. In all instances, the best fit u,v pairs indicate that an additional cohesive force (interpreted as hydrophobic in nature) is present within the thermophilic ortholog. The robustness of the observed cohesive force strongly substantiates the conclusions made herein regarding the improved hydrophobic packing of the thermophilic ortholog.
QSFR variability emerges through (subtle)differences within the H-bond network
The bacterial Periplasmic Binding Protein family
bPBP
ATP-binding domains
Transmembrane domains
Substrate
-- Extracellular --
-- Periplasm --
-- Cytosol --
Structure superposition of four bPBP homologs
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Local vs. global structural similarity
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Diversity within QSFR arises from subtledifferences within the H-bond network
R = 0.92R = -0.97
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Comparisons to apo vs. halo DSC experiments strongly supports the diversity observed within the mDCM results
apo-HBP
ligated-HBP
LAOBPGBP
mDCM results Kreimer et al. (2000), Eur J. Biochem.
Tm ~ 10KTm ~ 10K
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Backbone flexibility is predicted to be conserved
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Despite flexibility similarity along the backbone, cooperatively correlation varies significantly
HBP LAOBP GBP
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Diversity within QSFR characteristics arise fromsubtle differences within the H-bond network
HBP (red) vs. LAOBP (blue) HBP (red) vs. GBP (green)
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Similar amounts of (dis)similarity are observedwhen comparing open vs. closed conformations
Ligate
d (
close
d)
Apo (
open)
Fle
xibi
lity
inde
x
Livesay, et al. (2008). Chemistry Central Journal, 2:17.
Not quite science fiction: The ability to design a protein witha specific cooperative response may not be that far away…
Using this protocol, we designed a de novo hydrogen bond network…
A similar narrative is emerging from ourcomparisons of the thioredoxin family
Mottonen, et al., Proteins, in press.
The TRX family
Mottonen, et al., Proteins, in press.
Rigid cluster susceptibility
Mottonen, et al., Proteins, in press.
Relative locations of key points describing the mechanical vs. thermodynamic transitions
Mottonen, et al., Proteins, in press.
As with the bPBP family, backbone flexibility is mostly conserved across the TRX family
Mottonen, et al., Proteins, in press.
As with the bPBP family, backbone flexibility is mostly conserved across the TRX family
Mottonen, et al., Proteins, in press.
QSFR diversity is explained by comparing key pointsalong the mechanical vs. thermodynamic transition
Mottonen, et al., Proteins, in press.
The observed QSFR differences are consistent with the amount of differences within the H-bond network
Mottonen, et al., Proteins, in press.
TRXox vs. TRXred
QSFR response is a long-range effect
Mottonen, et al., Proteins, in press.
A similar narrative is emerging from ourcomparisons of the thioredoxin family
Periplasm(an oxidizing environment)
Cytosol(a reducing environment)
DsbAred
DsbAox
Trxred
Trxox
Mottonen, Livesay, Jacobs. In preparation.
The mDCM correctly predicts trends withinknown DsbA vs. Trx functional roles
Mottonen, Livesay, Jacobs. In preparation.