Spelling suggestions: "subject:"denatured state"" "subject:"renatured state""
1 |
UNDERSTANDING FORCES THAT CONTRIBUTE TO PROTEIN STABILITY: APPLICATION FOR INCREASING PROTEIN STABILITYFu, Hailong 2009 May 1900 (has links)
The aim of this study is to further our understanding of the forces that contribute
to protein stability and to investigate how site-directed mutagenesis might be used for
increasing protein stability. Eleven proteins ranging from 36 to 370 residues have been
studied here. A 36-residue VHP and a 337-residue VlsE were used as model systems for
studying the contribution of the hydrophobic effect on protein stability. Mutations were
made in both proteins which replaced bulky hydrophobic side chains with smaller ones.
All variants were less stable than their wild-type proteins. For VHP, the destabilizing
effects of mutations were smaller when compared with similar mutations reported in the
literature. For VlsE, a similarity was observed. This different behavior was investigated
and reconciled by the difference in hydrophobicity and cavity modeling for both
proteins. Therefore, the stabilizing mechanism of the hydrophobic effect appears to be
similar for both proteins.
Eight proteins were used as model systems for studying the effects of mutating
non-proline and non-glycine residues to statistically favored proline and glycine residues
in ?-turns. The results suggest that proline mutations generally increase protein stability, provided that the replaced residues are solvent exposed. The glycine mutations,
however, only have a stabilizing effect when the wild-type residues have ?, ? angles in
the L? region of Ramachandran plot. Nevertheless, this strategy still proves to be a
simple and efficient way for increasing protein stability.
Finally, using a combination of eight previously identified stabilizing mutations;
we successfully designed two RNase Sa variants (7S, 8S) that have both much higher
Tms and conformational stabilities than wild-type protein over the entire pH range
studied. Further studies of the heat capacity change upon unfolding (?Cps) for both
proteins and their variants suggest that residual structure may exist in the denatured state
of the 8S variant. An analysis of stability curves for both variants suggests that they
achieve their stabilization through different mechanisms, partly attributed to the different
role of their denatured states. The 7S variants may have a more rigid denatured state and
the 8S variant may have a compact denatured state in comparison with that of wild-type
RNase Sa.
|
2 |
The folding kinetics of ribonuclease Sa and a charge-reversal variantTrefethen, Jared M. 17 February 2005 (has links)
The primary objective was to study the kinetics of folding of RNase Sa. Wild-type
RNase Sa does not contain tryptophan. A tryptophan was substituted at residue
81 (WT*) to allow fluorescence spectroscopy to be used to monitor folding. This
tryptophan mutation did not change the stability. An analysis of the folding
kinetics of RNase Sa showed two folding phases, indicating the presence of an
intermediate and consistent with the following mechanism: D ↔ I ↔ N. Both
refolding limbs of the chevron plot (abcissa = final conc. of denaturant and
ordinate = kinetic rate) had non-zero slopes suggesting that proline
isomerization was not rate-limiting.
The conformational stability of a charge-reversed variant, WT*(D17R), of
a surface exposed residue on RNase Sa has been studied by equilibrium
techniques. This mutant with a single amino acid charge reversal of a surface
exposed residue resulted in decreased stability. Calculations using Coulombs
Law suggested that favorable electrostatic interactions in the denatured state
were the cause for the decreased stability for the charge-reversed variant.
Folding and unfolding kinetic studies were designed and conducted to study the
charge-reversal effect. Unfolding kinetics showed a 10-fold increase in the
unfolding rate constant for WT*(D17R) over WT* and no difference in the rate of
refolding.
Kinetics experiments were also conducted at pH 3 where protonation of
Asp17 (charge reversal site) would be expected to negate the observed kinetic
effect. At pH 3 the kinetics of unfolding of WT* RNase Sa and the WT*(D17R)
mutant were more similar. These kinetic results indicate that a single-site
charge reversal lowered the free energy of the denatured state as suspected.
Additionally, the results showed that the transition state was stabilized as well.
These results show that a specific Coulombic interaction lowered the free energy
in the denatured and transition state of the charge-reversal mutant, more than in
WT*. To our knowledge, this is the first demonstration that a favorable
electrostatic interaction in the denatured state ensemble has been shown to
influence the unfolding kinetics of a protein.
|
3 |
The effect of chirality and steric hindrance on intrinsic backbone conformational propensities: tools for protein designChilders, M.C., Towse, Clare-Louise, Daggett, V. 11 May 2016 (has links)
No / The conformational propensities of amino acids are an amalgamation of sequence effects, environmental effects and underlying intrinsic behavior. Many have attempted to investigate neighboring residue effects to aid in our understanding of protein folding and improve structure prediction efforts, especially with respect to difficult to characterize states, such as disordered or unfolded states. Host-guest peptide series are a useful tool in examining the propensities of the amino acids free from the surrounding protein structure. Here, we compare the distributions of the backbone dihedral angles (φ/ψ) of the 20 proteogenic amino acids in two different sequence contexts using the AAXAA and GGXGG host-guest pentapeptide series. We further examine their intrinsic behaviors across three environmental contexts: water at 298 K, water at 498 K, and 8 M urea at 298 K. The GGXGG systems provide the intrinsic amino acid propensities devoid of any conformational context. The alanine residues in the AAXAA series enforce backbone chirality, thereby providing a model of the intrinsic behavior of amino acids in a protein chain. Our results show modest differences in φ/ψ distributions due to the steric constraints of the Ala side chains, the magnitudes of which are dependent on the denaturing conditions. One of the strongest factors modulating φ/ψ distributions was the protonation of titratable side chains, and the largest differences observed were in the amino acid propensities for the rarely sampled αL region. / NIH
|
Page generated in 0.0837 seconds