5 research outputs found
Dispersion Forces and the Molecular Origin of Internal Friction in Protein
Internal
friction in macromolecules is one of the curious phenomena
that control conformational changes and reaction rates. It is held
here that dispersion interactions and London–van der Waals
forces between nonbonded atoms are major contributors to internal
friction. To demonstrate this, the flipping motion of aromatic rings
of F10 and Y97 amino acid residues of cytochrome <i>c</i> has been studied in glycerol/water mixtures by cross relaxation-suppressed
exchange nuclear magnetic resonance spectroscopy. The ring-flip rate
is highly overdamped by glycerol, but this is not due to the effect
of protein–solvent interactions on the Brownian dynamics of
the protein, because glycerol cannot penetrate into the protein to
slow the internal collective motions. Sound velocity in the protein
under matching solvent conditions shows that glycerol exerts its effect
by rather smothering the protein interior to produce reduced molecular
compressibility and root-mean-square volume fluctuation (δ<i>V</i><sub>RMS</sub>), implying an increased number of dispersion
interactions of nonbonded atoms. Hence, δ<i>V</i><sub>RMS</sub> can be used as a proxy for internal friction. By using
the ansatz that internal friction is related to nonbonded interactions
by the equation <i>f</i>(<i>n</i>) = <i>f</i><sub>0</sub> + <i>f</i><sub>1</sub><i>n</i> + <i>f</i><sub>2</sub><i>n</i><sup>2</sup> + ..., where
the variable <i>n</i> is the extent of nonbonded interactions
with <i>f</i><sub><i>i</i></sub> coefficients,
the barrier to aromatic ring rotation is found to be flat. Also interesting
is the appearance of a turnover region in the δ<i>V</i><sub>RMS</sub> dependence of the ring-flip rate, suggesting anomalous
internal diffusion. We conclude that cohesive forces among nonbonded
atoms are major contributors to the molecular origin of internal friction
Free Energy Landscape of Lysozyme: Multiple Near-Native Conformational States and Rollover in the Urea Dependence of Folding Energy
Deviation
from linearity of the equilibrium folding free energy
(Δ<i>G</i>) of proteins along the reaction coordinate
is scarcely known. Optical spectroscopic observables and NMR-measured
average molecular dimensional property of lysozyme with urea at pH
5 reveal that Δ<i>G</i> rolls over from linearity
under mild to strongly native-like conditions. The urea dependence
of Δ<i>G</i> is graphed in the 0–7 M range
of the denaturant by employing a series of guanidine hydrochloride
(GdnHCl)-induced equilibrium unfolding transitions, each in the presence
of a fixed level of urea. The observed linear dependence of Δ<i>G</i> on urea under denaturing conditions begins to deviate
as moderately native-like conditions are approached and eventually
rolls over under strongly native-like conditions. This is atypical
of the upward curvature in the Δ<i>G</i> vs denaturant
plot predicted by the denaturant binding model. On increasing the
denaturant concentration from 0 to 5 M, the hydrodynamic radius of
lysozyme shrinks by ∼2 Å. We suggest subdenaturing levels
of urea affect the population distribution among multiple near-native
isoenergetic conformational states so as to promote them sequentially
with increments of the denaturant. We use a multiple-state sequential
model to show that the keel over of Δ<i>G</i> occurs
due to these near-native alternative states in the native ensemble
used for defining the unfolding equilibrium constant (<i>K</i><sub>U</sub>), which we assume to vary linearly with urea. The results
and the model appear to indicate a rugged flat bottom in the free
energy landscape wherein population distribution of native-like states
is modulated by urea-affected interstate motions
Expansion and Internal Friction in Unfolded Protein Chain
Similarities
in global properties of homopolymers and unfolded
proteins provide approaches to mechanistic description of protein
folding. Here, hydrodynamic properties and relaxation rates of the
unfolded state of carbonmonoxide-liganded cytochrome <i>c</i> (cyt-CO) have been measured using nuclear magnetic resonance and
laser photolysis methods. Hydrodynamic radius of the unfolded chain
gradually increases as the solvent turns increasingly better, consistent
with theory. Curiously, however, the rate of intrachain contact formation
also increases with an increasing denaturant concentration, which,
by Szabo, Schulten, and Schulten theory for the rate of intramolecular
contact formation in a Gaussian polymer, indicates growing intramolecular
diffusion. It is argued that diminishing nonbonded atom interactions
with increasing denaturant reduces internal friction and, thus, increases
the rate of polypeptide relaxation. Qualitative scaling of the extent
of unfolding with nonbonded repulsions allows for description of internal
friction by a phenomenological model. The degree of nonbonded atom
interactions largely determines the extent of internal friction
Solution NMR Structure and Backbone Dynamics of Partially Disordered <i>Arabidopsis thaliana</i> Phloem Protein 16-1, a Putative mRNA Transporter
Although
RNA-binding proteins in plant phloem are believed to perform
long-distance systemic transport of RNA in the phloem conduit, the
structure of none of them is known. <i>Arabidopsis thaliana</i> phloem protein 16-1 (<i>At</i>PP16-1) is such a putative
mRNA transporter whose structure and backbone dynamics have been studied
at pH 4.1 and 25 °C by high-resolution nuclear magnetic resonance
spectroscopy. Results obtained using basic optical spectroscopic tools
show that the protein is unstable with little secondary structure
near the physiological pH of the phloem sap. Fluorescence-monitored
titrations reveal that <i>At</i>PP16-1 binds not only <i>A</i>. <i>thaliana</i> RNA (<i>K</i><sub>diss</sub> ∼ 67 nM) but also sheared DNA and model dodecamer
DNA, though the affinity for DNA is ∼15-fold lower. In the
solution structure of the protein, secondary structural elements are
formed by residues 3–9 (β1), 56–62 (β2),
133–135 (β3), and 96–110 (α-helix). Most
of the rest of the chain segments are disordered. The N-terminally
disordered regions (residues 10–55) form a small lobe, which
conjoins the rest of the molecule via a deep and large irregular cleft
that could have functional implications. The average order parameter
extracted by model-free analysis of <sup>15</sup>N relaxation and
{<sup>1</sup>H}–<sup>15</sup>N heteronuclear NOE data is 0.66,
suggesting less restricted backbone motion. The average conformational
entropy of the backbone NH vectors is −0.31 cal mol<sup>–1</sup> K<sup>–1</sup>. These results also suggest structural disorder
in <i>At</i>PP16-1
Unfolding Action of Alcohols on a Highly Negatively Charged State of Cytochrome <i>c</i>
It is well-known that hydrophobic effect play a major
role in alcohol–protein interactions leading to structure unfolding.
Studies with extremely alkaline cytochrome <i>c</i> (U<sub>B</sub> state, pH 13) in the presence of the first four alkyl alcohols
suggests that the hydrophobic effect persistently overrides even though
the protein carries a net charge of −17 under these conditions.
Equilibrium unfolding of the U<sub>B</sub> state is accompanied by
an unusual expansion of the chain involving an intermediate, I<sub>alc</sub>, from which water is preferentially excluded, the extent
of water exclusion being greater with the hydrocarbon content of the
alcohol. The mobility and environmental averaging of side chains in
the I<sub>alc</sub> state are generally constrained relative to those
in the U<sub>B</sub> state. A few nuclear magnetic resonance-detected
tertiary interactions are also found in the I<sub>alc</sub> state.
The fact that the I<sub>alc</sub> state populates at low concentrations
of methanol and ethanol and the fact that the extent of chain expansion
in this state approaches that of the U<sub>B</sub> state indicate
a definite influence of electrostatic repulsion severed by the low
dielectric of the water/alcohol mixture. Interestingly, the U<sub>B</sub> ⇌ I<sub>alc</sub> segment of the U<sub>B</sub> ⇌
I<sub>alc</sub> ⇌ U equilibrium, where U is the unfolded state,
accounts for roughly 85% of the total number of water molecules preferentially
excluded in unfolding. Stopped-flow refolding results report on a
submillisecond hydrophobic collapse during which almost the entire
buried surface area associated with the U<sub>B</sub> state is recovered,
suggesting the overwhelming
influence of hydrophobic interaction over electrostatic repulsions