21 research outputs found
Development of CHARMM-Compatible Force-Field Parameters for Cobalamin and Related Cofactors from Quantum Mechanical Calculations
Corrinoid
cofactors such as cobalamin are used by many enzymes
and are essential for most living organisms. Therefore, there is broad
interest in investigating cobalamināprotein interactions with
molecular dynamics simulations. Previously developed parameters for
cobalamins are based mainly on crystal structure data. Here, we report
CHARMM-compatible force field parameters for several corrinoids developed
from quantum mechanical calculations. We provide parameters for corrinoids
in three oxidation states, Co<sup>3+</sup>, Co<sup>2+</sup>, and Co<sup>1+</sup>, and with various axial ligands. Lennard-Jones parameters
for the cobalt center in the CoĀ(II) and CoĀ(I) states were optimized
using a helium atom probe, and partial atomic charges were obtained
with a combination of natural population analysis (NPA) and restrained
electrostatic potential (RESP) fitting approaches. The Force Field
Toolkit was used to optimize all bonded terms. The resulting parameters,
determined solely from calculations of cobalamin alone or in water,
were then validated by assessing their agreement with density functional
theory geometries and by analyzing molecular dynamics simulation trajectories
of several corrinoid proteins for which X-ray crystal structures are
available. In each case, we obtained excellent agreement with the
reference data. In comparison to previous CHARMM-compatible parameters
for cobalamin, we observe a better agreement for the fold angle and
lower RMSD in the cobalamin binding site. The approach described here
is readily adaptable for developing CHARMM-compatible force-field
parameters for other corrinoids or large biomolecules
Toward Quantitatively Accurate Calculation of the Redox-Associated AcidāBase and Ligand Binding Equilibria of Aquacobalamin
Redox processes in complex transition
metal-containing species
are often intimately associated with changes in ligand protonation
states and metal coordination number. A major challenge is therefore
to develop consistent computational approaches for computing pH-dependent
redox and ligand dissociation properties of organometallic species.
Reduction of the Co center in the vitamin B12 derivative aquacobalamin
can be accompanied by ligand dissociation, protonation, or both, making
these properties difficult to compute accurately. We examine this
challenge here by using density functional theory and continuum solvation
to compute Coāligand binding equilibrium constants (<i>K</i><sub>on/off</sub>), p<i>K</i><sub>a</sub>s, and
reduction potentials for models of aquacobalamin in aqueous solution.
We consider two models for cobalamin ligand coordination: the first
follows the hexa, penta, tetra coordination scheme for Co<sup>III</sup>, Co<sup>II</sup>, and Co<sup>I</sup> species, respectively, and
the second model features saturation of each vacant axial coordination
site on Co<sup>II</sup> and Co<sup>I</sup> species with a single,
explicit water molecule to maintain six directly interacting ligands
or water molecules in each oxidation state. Comparing these two coordination
schemes in combination with five dispersion-corrected density functionals,
we find that the accuracy of the computed properties is largely independent
of the scheme used, but including only a continuum representation
of the solvent yields marginally better results than saturating the
first solvation shell around Co throughout. PBE performs best, displaying
balanced accuracy and superior performance overall, with RMS errors
of 80 mV for seven reduction potentials, 2.0 log units for five p<i>K</i><sub>a</sub>s and 2.3 log units for two log <i>K</i><sub>on/off</sub> values for the aquacobalamin system. Furthermore,
we find that the BP86 functional commonly used in corrinoid studies
suffers from erratic behavior and inaccurate descriptions of Coāaxial
ligand binding, leading to substantial errors in predicted p<i>K</i><sub>a</sub>s and <i>K</i><sub>on/off</sub> values.
These findings demonstrate the effectiveness of the present approach
for computing electrochemical and thermodynamic properties of a complex
transition metal-containing cofactor
Quantum Chemical Calculation of p<i>K</i><sub>a</sub>s of Environmentally Relevant Functional Groups: Carboxylic Acids, Amines, and Thiols in Aqueous Solution
Developing
accurate quantum chemical approaches for calculating
p<i>K</i><sub>a</sub>s is of broad interest. Useful accuracy
can be obtained by using density functional theory (DFT) in combination
with a polarizable continuum solvent model. However, some classes
of molecules present problems for this approach, yielding errors greater
than 5 p<i>K</i> units. Various methods have been developed
to improve the accuracy of the combined strategy. These methods perform
well but either do not generalize or introduce additional degrees
of freedom, increasing the computational cost. The Solvation Model
based on Density (SMD) has emerged as one of the most commonly used
continuum solvent models. Nevertheless, for some classes of organic
compounds, e.g., thiols, the p<i>K</i><sub>a</sub>s calculated
with the original SMD model show errors of 6ā10 p<i>K</i> units, and we traced these errors to inaccuracies in the solvation
free energies of the anions. To improve the accuracy of p<i>K</i><sub>a</sub>s calculated with DFT and the SMD model, we developed
a scaled solvent-accessible surface approach for constructing the
soluteāsolvent boundary. By using a ādirectā
approach, in which all quantities are computed in the presence of
the continuum solvent, the use of thermodynamic cycles is avoided.
Furthermore, no explicit water molecules are required. Three benchmark
data sets of experimentally measured p<i>K</i><sub>a</sub> values, including 28 carboxylic acids, 10 aliphatic amines, and
45 thiols, were used to assess the optimized SMD model, which we call
SMD with a scaled solvent-accessible surface (SMD<sub>sSAS</sub>).
Of the methods tested, the M06-2X density functional approximation,
6-31+GĀ(d,p) basis set, and SMD<sub>sSAS</sub> solvent model provided
the most accurate p<i>K</i><sub>a</sub>s for each set, yielding
mean unsigned errors of 0.9, 0.4, and 0.5 p<i>K</i> units,
respectively, for carboxylic acids, aliphatic amines, and thiols.
This approach is therefore useful for efficiently calculating the
p<i>K</i><sub>a</sub>s of environmentally relevant functional
groups
Hydrolysis of DFP and the Nerve Agent (<i>S</i>)āSarin by DFPase Proceeds along Two Different Reaction Pathways: Implications for Engineering Bioscavengers
Organophosphorus
(OP) nerve agents such as (<i>S</i>)-sarin
are among the most highly toxic compounds that have been synthesized.
Engineering enzymes that catalyze the hydrolysis of nerve agents (ābioscavengersā)
is an emerging prophylactic approach to diminish their toxic effects.
Although its native function is not known, diisopropyl fluorophosphatase
(DFPase) from Loligo vulgaris catalyzes
the hydrolysis of OP compounds. Here, we investigate the mechanisms
of diisopropylfluorophosphate (DFP) and (<i>S</i>)-sarin
hydrolysis by DFPase with quantum mechanical/molecular mechanical
umbrella sampling simulations. We find that the mechanism for hydrolysis
of DFP involves nucleophilic attack by Asp229 on phosphorus to form
a pentavalent intermediate. PāF bond dissociation then yields
a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations
suggest that a water molecule, coordinated to the catalytic Ca<sup>2+</sup>, donates a proton to Asp121 and then attacks the tetrahedral
phosphoacyl intermediate to liberate the diisopropylphosphate product.
In contrast, the calculated free energy barrier for hydrolysis of
(<i>S</i>)-sarin by the same mechanism is highly unfavorable,
primarily because of the instability of the pentavalent phosphoenzyme
species. Instead, simulations suggest that hydrolysis of (<i>S</i>)-sarin proceeds by a mechanism in which Asp229 could activate
an intervening water molecule for nucleophilic attack on the substrate.
These findings may lead to improved strategies for engineering DFPase
and related six-bladed Ī²-propeller folds for more efficient
degradation of OP compounds
Exploring Covalent Allosteric Inhibition of Antigen 85C from <i>Mycobacterium tuberculosis</i> by Ebselen Derivatives
Previous
studies identified ebselen as a potent <i>in vitro</i> and <i>in vivo</i> inhibitor of the <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) antigen 85 (Ag85) complex, comprising three
homologous enzymes required for the biosynthesis of the mycobacterial
cell wall. In this study, the <i>Mtb</i> Ag85C enzyme was
cocrystallized with azido and adamantyl ebselen derivatives, resulting
in two crystallographic structures of 2.01 and 1.30 Ć
resolution,
respectively. Both structures displayed the anticipated covalent modification
of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide
bond. Continuous difference density for both thiol modifiers allowed
for the assessment of interactions that influence ebselen binding
and inhibitor orientation that were unobserved in previous Ag85C ebselen
structures. The <i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> values for ebselen, adamantyl ebselen, and azido ebselen
support the importance of observed constructive chemical interactions
with Arg239 for increased <i>in vitro</i> efficacy toward
Ag85C. To better understand the <i>in vitro</i> kinetic
properties of these ebselen derivatives, the energetics of specific
proteināinhibitor interactions and relative reaction free energies
were calculated for ebselen and both derivatives using density functional
theory. These studies further support the different <i>in vitro</i> properties of ebselen and two select ebselen derivatives from our
previously published ebselen library with respect to kinetics and
proteināinhibitor interactions. In both structures, the Ī±9
helix was displaced farther from the enzyme active site than the previous
Ag85C ebselen structure, resulting in the restructuring of a connecting
loop and imparting a conformational change to residues believed to
play a role in substrate binding specific to Ag85C. These notable
structural changes directly affect protein stability, reducing the
overall melting temperature by up to 14.5 Ā°C, resulting in the
unfolding of protein at physiological temperatures. Additionally,
this structural rearrangement due to covalent allosteric modification
creates a sizable solvent network that encompasses the active site
and extends to the modified Cys209 residue. In all, this study outlines
factors that influence enzyme inhibition by ebselen and its derivatives
while further highlighting the effects of the covalent modification
of Cys209 by said inhibitors on the structure and stability of Ag85C.
Furthermore, the results suggest a strategy for developing new classes
of Ag85 inhibitors with increased specificity and potency
Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LCāMS/MS Data
Redox proteomics has yielded molecular
insight into diseases of
protein dysfunction attributable to oxidative stress, underscoring
the need for robust detection of protein oxidation products. Additionally,
oxidative protein surface mapping techniques utilize hydroxyl radicals
to gain structural insight about solvent exposure. Interpretation
of tandem mass spectral data is a critical challenge for such investigations,
because reactive oxygen species target a wide breadth of amino acids.
Additionally, oxidized peptides may be generated in a wide range of
abundances since the reactivity of hydroxyl radicals with different
amino acids spans 3 orders of magnitude. Taken together, these attributes
of oxidative footprinting pose both experimental and computational
challenges to detecting oxidized peptides that are naturally less
abundant than their unoxidized counterparts. In this study, model
proteins were oxidized electrochemically and analyzed at both the
intact protein and peptide levels. A multidimensional chromatographic
strategy was utilized to expand the dynamic range of oxidized peptide
measurements. Peptide mass spectral data were searched by the āhybridā
software packages Inspect and Byonic, which incorporate <i>de
novo</i> elements of spectral interpretation into a database
search. This dynamic search capacity accommodates the challenge of
searching for more than 40 oxidative mass shifts that can occur in
a staggering variety of possible combinatorial occurrences. A prevailing
set of oxidized residues was identified with this comparative approach,
and evaluation of these sites was informed by solvent accessible surface
area gleaned through molecular dynamics simulations. Along with increased
levels of oxidation around highly reactive āhotspotā
sites as expected, the enhanced sensitivity of these measurements
uncovered a surprising level of oxidation on less reactive residues
Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LCāMS/MS Data
Redox proteomics has yielded molecular
insight into diseases of
protein dysfunction attributable to oxidative stress, underscoring
the need for robust detection of protein oxidation products. Additionally,
oxidative protein surface mapping techniques utilize hydroxyl radicals
to gain structural insight about solvent exposure. Interpretation
of tandem mass spectral data is a critical challenge for such investigations,
because reactive oxygen species target a wide breadth of amino acids.
Additionally, oxidized peptides may be generated in a wide range of
abundances since the reactivity of hydroxyl radicals with different
amino acids spans 3 orders of magnitude. Taken together, these attributes
of oxidative footprinting pose both experimental and computational
challenges to detecting oxidized peptides that are naturally less
abundant than their unoxidized counterparts. In this study, model
proteins were oxidized electrochemically and analyzed at both the
intact protein and peptide levels. A multidimensional chromatographic
strategy was utilized to expand the dynamic range of oxidized peptide
measurements. Peptide mass spectral data were searched by the āhybridā
software packages Inspect and Byonic, which incorporate <i>de
novo</i> elements of spectral interpretation into a database
search. This dynamic search capacity accommodates the challenge of
searching for more than 40 oxidative mass shifts that can occur in
a staggering variety of possible combinatorial occurrences. A prevailing
set of oxidized residues was identified with this comparative approach,
and evaluation of these sites was informed by solvent accessible surface
area gleaned through molecular dynamics simulations. Along with increased
levels of oxidation around highly reactive āhotspotā
sites as expected, the enhanced sensitivity of these measurements
uncovered a surprising level of oxidation on less reactive residues
Exploring Covalent Allosteric Inhibition of Antigen 85C from <i>Mycobacterium tuberculosis</i> by Ebselen Derivatives
Previous
studies identified ebselen as a potent <i>in vitro</i> and <i>in vivo</i> inhibitor of the <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) antigen 85 (Ag85) complex, comprising three
homologous enzymes required for the biosynthesis of the mycobacterial
cell wall. In this study, the <i>Mtb</i> Ag85C enzyme was
cocrystallized with azido and adamantyl ebselen derivatives, resulting
in two crystallographic structures of 2.01 and 1.30 Ć
resolution,
respectively. Both structures displayed the anticipated covalent modification
of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide
bond. Continuous difference density for both thiol modifiers allowed
for the assessment of interactions that influence ebselen binding
and inhibitor orientation that were unobserved in previous Ag85C ebselen
structures. The <i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> values for ebselen, adamantyl ebselen, and azido ebselen
support the importance of observed constructive chemical interactions
with Arg239 for increased <i>in vitro</i> efficacy toward
Ag85C. To better understand the <i>in vitro</i> kinetic
properties of these ebselen derivatives, the energetics of specific
proteināinhibitor interactions and relative reaction free energies
were calculated for ebselen and both derivatives using density functional
theory. These studies further support the different <i>in vitro</i> properties of ebselen and two select ebselen derivatives from our
previously published ebselen library with respect to kinetics and
proteināinhibitor interactions. In both structures, the Ī±9
helix was displaced farther from the enzyme active site than the previous
Ag85C ebselen structure, resulting in the restructuring of a connecting
loop and imparting a conformational change to residues believed to
play a role in substrate binding specific to Ag85C. These notable
structural changes directly affect protein stability, reducing the
overall melting temperature by up to 14.5 Ā°C, resulting in the
unfolding of protein at physiological temperatures. Additionally,
this structural rearrangement due to covalent allosteric modification
creates a sizable solvent network that encompasses the active site
and extends to the modified Cys209 residue. In all, this study outlines
factors that influence enzyme inhibition by ebselen and its derivatives
while further highlighting the effects of the covalent modification
of Cys209 by said inhibitors on the structure and stability of Ag85C.
Furthermore, the results suggest a strategy for developing new classes
of Ag85 inhibitors with increased specificity and potency
Cluster-Continuum Calculations of Hydration Free Energies of Anions and Group 12 Divalent Cations
Understanding aqueous phase processes involving group
12 metal
cations is relevant to both environmental and biological sciences.
Here, quantum chemical methods and polarizable continuum models are
used to compute the hydration free energies of a series of divalent
group 12 metal cations (Zn<sup>2+</sup>, Cd<sup>2+</sup>, and Hg<sup>2+</sup>) together with Cu<sup>2+</sup> and the anions OH<sup>ā</sup>, SH<sup>ā</sup>, Cl<sup>ā</sup>, and F<sup>ā</sup>. A cluster-continuum method is employed, in which gas-phase clusters
of the ion and explicit solvent molecules are immersed in a dielectric
continuum. Two approaches to define the size of the soluteāwater
cluster are compared, in which the number of explicit waters used
is either held constant or determined variationally as that of the
most favorable hydration free energy. Results obtained with various
polarizable continuum models are also presented. Each leg of the relevant
thermodynamic cycle is analyzed in detail to determine how different
terms contribute to the observed mean signed error (MSE) and the standard
deviation of the error (STDEV) between theory and experiment. The
use of a constant number of water molecules for each set of ions is
found to lead to predicted relative trends that benefit from error
cancellation. Overall, the best results are obtained with MP2 and
the Solvent Model D polarizable continuum model (SMD), with eight
explicit water molecules for anions and 10 for the metal cations,
yielding a STDEV of 2.3 kcal mol<sup>ā1</sup> and MSE of 0.9
kcal mol<sup>ā1</sup> between theoretical and experimental
hydration free energies, which range from ā72.4 kcal mol<sup>ā1</sup> for SH<sup>ā</sup> to ā505.9 kcal mol<sup>ā1</sup> for Cu<sup>2+</sup>. Using B3PW91 with DFT-D3 dispersion
corrections (B3PW91-D) and SMD yields a STDEV of 3.3 kcal mol<sup>ā1</sup> and MSE of 1.6 kcal mol<sup>ā1</sup>, to which
adding MP2 corrections from smaller divalent metal cation water molecule
clusters yields very good agreement with the full MP2 results. Using
B3PW91-D and SMD, with two explicit water molecules for anions and
six for divalent metal cations, also yields reasonable agreement with
experimental values, due in part to fortuitous error cancellation
associated with the metal cations. Overall, the results indicate that
the careful application of quantum chemical cluster-continuum methods
provides valuable insight into aqueous ionic processes that depend
on both local and long-range electrostatic interactions with the solvent
Cluster-Continuum Calculations of Hydration Free Energies of Anions and Group 12 Divalent Cations
Understanding aqueous phase processes involving group
12 metal
cations is relevant to both environmental and biological sciences.
Here, quantum chemical methods and polarizable continuum models are
used to compute the hydration free energies of a series of divalent
group 12 metal cations (Zn<sup>2+</sup>, Cd<sup>2+</sup>, and Hg<sup>2+</sup>) together with Cu<sup>2+</sup> and the anions OH<sup>ā</sup>, SH<sup>ā</sup>, Cl<sup>ā</sup>, and F<sup>ā</sup>. A cluster-continuum method is employed, in which gas-phase clusters
of the ion and explicit solvent molecules are immersed in a dielectric
continuum. Two approaches to define the size of the soluteāwater
cluster are compared, in which the number of explicit waters used
is either held constant or determined variationally as that of the
most favorable hydration free energy. Results obtained with various
polarizable continuum models are also presented. Each leg of the relevant
thermodynamic cycle is analyzed in detail to determine how different
terms contribute to the observed mean signed error (MSE) and the standard
deviation of the error (STDEV) between theory and experiment. The
use of a constant number of water molecules for each set of ions is
found to lead to predicted relative trends that benefit from error
cancellation. Overall, the best results are obtained with MP2 and
the Solvent Model D polarizable continuum model (SMD), with eight
explicit water molecules for anions and 10 for the metal cations,
yielding a STDEV of 2.3 kcal mol<sup>ā1</sup> and MSE of 0.9
kcal mol<sup>ā1</sup> between theoretical and experimental
hydration free energies, which range from ā72.4 kcal mol<sup>ā1</sup> for SH<sup>ā</sup> to ā505.9 kcal mol<sup>ā1</sup> for Cu<sup>2+</sup>. Using B3PW91 with DFT-D3 dispersion
corrections (B3PW91-D) and SMD yields a STDEV of 3.3 kcal mol<sup>ā1</sup> and MSE of 1.6 kcal mol<sup>ā1</sup>, to which
adding MP2 corrections from smaller divalent metal cation water molecule
clusters yields very good agreement with the full MP2 results. Using
B3PW91-D and SMD, with two explicit water molecules for anions and
six for divalent metal cations, also yields reasonable agreement with
experimental values, due in part to fortuitous error cancellation
associated with the metal cations. Overall, the results indicate that
the careful application of quantum chemical cluster-continuum methods
provides valuable insight into aqueous ionic processes that depend
on both local and long-range electrostatic interactions with the solvent