Membrane Catalyzed Formation of Nucleotide Clusters and Their Role in the Origins of Life: Insights from Molecular Simulations and Lattice Modeling.

One of the mysteries in studying the molecular "Origin of Life" is the emergence of RNA and RNA-based life forms, where nonenzymatic polymerization of nucleotides is a crucial hypothesis in formation of large RNA chains. The nonenzymatic polymerization can be mediated by various environmental settings, such as cycles of hydration and dehydration, temperature variations, and proximity to a variety of organizing matrices, such as clay, salt, fatty acids, lipid membrane, and mineral surface. In this work, we explore the influence of different phases of the lipid membrane toward nucleotide organization and polymerization in a simulated prebiotic setting. Our molecular simulations quantify the localization propensity of a mononucleotide, uridine monophosphate (UMP), in distinct membrane settings. We perform all-atom molecular dynamics (MD) simulations to estimate the role of the monophasic and biphasic membranes in modifying the behavior of UMPs localization and their clustering mechanism. Based on the interaction energy of mononucleotides with the membrane and their diffusion profile from our MD calculations, we developed a lattice-based model to explore the thermodynamic limits of the observations made from the MD simulations. The mathematical model substantiates our hypothesis that the lipid layers can act as unique substrates for "catalyzing" polymerization of mononucleotides due to the inherent spatiotemporal heterogeneity and phase change behavior.

Parallel triplet formation pathways in a singlet fission material.

Harvesting long-lived free triplets in high yields by utilizing organic singlet fission materials can be the cornerstone for increasing photovoltaic efficiencies potentially. However, except for polyacenes, which are the most studied systems in the singlet fission field, spin-entangled correlated triplet pairs and free triplets born through singlet fission are relatively poorly characterized. By utilizing transient absorption and photoluminescence spectroscopy in supramolecular aggregate thin films consisting of Hamilton-receptor-substituted diketopyrrolopyrrole derivatives, we show that photoexcitation gives rise to the formation of spin-0 correlated triplet pair 1(TT) from the lower Frenkel exciton state. The existence of 1(TT) is proved through faint Herzberg-Teller emission that is enabled by vibronic coupling and correlated with an artifact-free triplet-state photoinduced absorption in the near-infrared. Surprisingly, transient electron paramagnetic resonance reveals that long-lived triplets are produced through classical intersystem crossing instead of 1(TT) dissociation, with the two pathways in competition. Moreover, comparison of the triplet-formation dynamics in J-like and H-like thin films with the same energetics reveals that spin-orbit coupling mediated intersystem crossing persists in both. However, 1(TT) only forms in the J-like film, pinpointing the huge impact of intermolecular coupling geometry on singlet fission dynamics.

Sequence dependent folding motifs of the secondary structures of Gly-Pro and Pro-Gly containing oligopeptides.

Folding motifs of the secondary structures of peptides and proteins are primarily based on the hydrogen bonding interactions in the backbone as well as the sequence of the amino acid residues present. For instance, the ß-turn structure directed by the Pro-Gly sequence is the key to the ß-hairpin structure of peptides/proteins as well as a selective site for the enzymatic hydroxylation of pro-collagen. Herein, we have investigated the sequence dependent folding motifs of end-protected Gly-Pro and Pro-Gly dipeptides using a combination of gas phase laser spectroscopy, quantum chemistry calculations, solution phase IR and NMR spectroscopy and single crystal X-Ray diffraction (XRD). All three observed conformers of the Gly-Pro peptide in the gas phase have been found to have extended ß-strand or polyproline-II (PP-II) structures with C5-C7 hydrogen bonding interactions, which correlates well with the structure obtained from solution phase spectroscopy and XRD. On the other hand, we have found that the Pro-Gly peptide has a C10/ß-turn structure in the solution phase in contrast to the C7-C7 (i.e. 27-ribbon) structure observed in the gas phase. Although the lowest energy structure in the gas phase is not C10, we find that C7-C7 is an abundantly found structural motif of Pro-Gly containing peptides in the Cambridge Structural Database, indicating that the gas phase conformers are not sampling any unusual forms. We surmise that the role of the solvent could be crucial in dictating the preferential stabilization of the C10 structure in the solution phase. The present investigation provides a comprehensive picture of the folding motifs of the Gly-Pro and Pro-Gly peptides observed in the gas phase and condensed phase weaving a fine interplay of the intrinsic conformational properties, solvation, and crystal packing of the peptides.

Cross-Talk between Overlap Interactions in Biomolecules: A Case Study of the ß-Turn Motif.

Noncovalent interactions play a pivotal role in regulating protein conformation, stability and dynamics. Among the quantum mechanical (QM) overlap-based noncovalent interactions, n→π* is the best understood with studies ranging from small molecules to ß-turns of model proteins such as GB1. However, these investigations do not explore the interplay between multiple overlap interactions in contributing to local structure and stability. In this work, we identify and characterize all noncovalent overlap interactions in the ß-turn, an important secondary structural element that facilitates the folding of a polypeptide chain. Invoking a QM framework of natural bond orbitals, we demonstrate the role of several additional interactions such as n→σ* and π→π* that are energetically comparable to or larger than n→π*. We find that these interactions are sensitive to changes in the side chain of the residues in the ß-turn of GB1, suggesting that the n→π* may not be the only component in dictating ß-turn conformation and stability. Furthermore, a database search of n→σ* and π→π* in the PDB reveals that they are prevalent in most proteins and have significant interaction energies (∼1 kcal/mol). This indicates that all overlap interactions must be taken into account to obtain a comprehensive picture of their contributions to protein structure and energetics. Lastly, based on the extent of QM overlaps and interaction energies, we propose geometric criteria using which these additional interactions can be efficiently tracked in broad database searches.

High resolution ensemble description of metamorphic and intrinsically disordered proteins using an efficient hybrid parallel tempering scheme.

Mapping free energy landscapes of complex multi-funneled metamorphic proteins and weakly-funneled intrinsically disordered proteins (IDPs) remains challenging. While rare-event sampling molecular dynamics simulations can be useful, they often need to either impose restraints or reweigh the generated data to match experiments. Here, we present a parallel-tempering method that takes advantage of accelerated water dynamics and allows efficient and accurate conformational sampling across a wide variety of proteins. We demonstrate the improved sampling efficiency by benchmarking against standard model systems such as alanine di-peptide, TRP-cage and ß-hairpin. The method successfully scales to large metamorphic proteins such as RFA-H and to highly disordered IDPs such as Histatin-5. Across the diverse proteins, the calculated ensemble averages match well with the NMR, SAXS and other biophysical experiments without the need to reweigh. By allowing accurate sampling across different landscapes, the method opens doors for sampling free energy landscape of complex uncharted proteins.

Double Domain Swapping in Human γC and γD Crystallin Drives Early Stages of Aggregation.

Human γD (HγD) and γC (HγC) are two-domain crystallin (Crys) proteins expressed in the nucleus of the eye lens. Structural perturbations in the protein often trigger aggregation, which eventually leads to cataract. To decipher the underlying molecular mechanism, it is important to characterize the partially unfolded conformations, which are aggregation-prone. Using a coarse grained protein model and molecular dynamics simulations, we studied the role of on-pathway folding intermediates in the early stages of aggregation. The multidimensional free energy surface revealed at least three different folding pathways with the population of partially structured intermediates. The two dominant pathways confirm sequential folding of the N-terminal [Ntd] and the C-terminal domains [Ctd], while the third, least favored, pathway involves intermediates where both the domains are partially folded. A native-like intermediate (I*), featuring the folded domains and disrupted interdomain contacts, gets populated in all three pathways. I* forms domain swapped dimers by swapping the entire Ntds and Ctds with other monomers. Population of such oligomers can explain the increased resistance to unfolding resulting in hysteresis observed in the folding experiments of HγD Crys. An ensemble of double domain swapped dimers are also formed during refolding, where intermediates consisting of partially folded Ntds and Ctds swap secondary structures with other monomers. The double domain swapping model presented in our study provides structural insights into the early events of aggregation in Crys proteins and identifies the key secondary structural swapping elements, where introducing mutations will aid in regulating the overall aggregation propensity.

Increasing protein stability by engineering the n → π* interaction at the ß-turn.

Abundant n → π* interactions between adjacent backbone carbonyl groups, identified by statistical analysis of protein structures, are predicted to play an important role in dictating the structure of proteins. However, experimentally testing the prediction in proteins has been challenging due to the weak nature of this interaction. By amplifying the strength of the n → π* interaction via amino acid substitution and thioamide incorporation at a solvent exposed ß-turn within the GB1 proteins and Pin 1 WW domain, we demonstrate that an n → π* interaction increases the structural stability of proteins by restricting the ϕ torsion angle. Our results also suggest that amino acid side-chain identity and its rotameric conformation play an important and decisive role in dictating the strength of an n → π* interaction.

Deuterium isotope effect in fluorescence of gaseous oxazine dyes.

The increased utility of fluorescence-based methods in recent years has highlighted the need for brighter, more efficient fluorophores. In order to design these fluorophores, an improved fundamental understanding is necessary of the structural components that intrinsically effect fluorescence efficiency. Here, we characterize the intrinsic effects of deuteration on fluorescence from gaseous oxazine dyes, without the influence of dye-solvent interactions, by making use of an ion trap mass spectrometer that has been altered to enable optical measurements. Comparison of emission spectra of four oxazine dyes: cresyl violet, oxazine 4, oxazine 170, and darrow red, show little change in profile upon deuteration of amine groups. However, deuteration significantly increases the efficiency of fluorescence with an increase in fluorescence lifetime and brightness by 10-23% for the gaseous dyes. This increase is less than half that of the quantum yield increase observed in deuterated solution. This indicates the large fluorescence efficiency changes for the oxazine dyes in deuterated solution result from a combination of both intrinsic effects as well as substantial contribution from altered fluorophore-solvent interactions. The intrinsic effects behind increased lifetime upon deuteration are explored using time-dependent density functional theory (TD-DFT) calculations of potential energy surfaces (PESs) for ground and low lying excited electronic states. In accord with experimental observations, calculated S1-S0 emission spectra show only minor differences between deuterated and non-deuterated forms indicating that the deuteration does not affect the radiative channel appreciably. Relaxed PES scans along the torsional motions of the amino groups reveal that the increase in lifetimes upon deuteration is likely due to quenching of different radiationless changes channels in different oxazine dyes. Calculations suggest that tunneling to access twisted intramolecular charge transfer states in S1 is critical in several of the oxazines. However, in at least one of the dyes examined, the large isotope effect is more likely due to differences in intersystem crossing rates. Overall, this combined experimental and computational investigation elucidates the photophysics of a well-known fluorescent scaffold and provides insight into how small differences can dramatically affect fluorescence outcomes.

Effects of maturation on the conformational free-energy landscape of SOD1.

Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer's, Parkinson's, and Huntington's diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components. We have shown previously that immature SOD1, lacking bound Cu and Zn metal ions and the intrasubunit disulfide bond (apoSOD12SH), has a rugged free-energy surface (FES) and exchanges with four other conformations (excited states) that have millisecond lifetimes and sparse populations on the order of a few percent. Here, we examine further states of SOD1 along its maturation pathway, as well as those off-pathway resulting from metal loss that have been observed in proteinaceous inclusions. Metallation and disulfide bond formation lead to structural transformations including local ordering of the electrostatic loop and native dimerization that are observed in rare conformers of apoSOD12SH; thus, SOD1 maturation may occur via a population-switch mechanism whereby posttranslational modifications select for preexisting structures on the FES. Metallation and oxidation of SOD1 stabilize the native, mature conformation and decrease the number of detected excited conformational states, suggesting that it is the immature forms of the protein that contribute to misfolded conformations in vivo rather than the highly stable enzymatically active dimer.

Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data.

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes.

Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles.

The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state.

New Insights into the State Trapping of UV-Excited Thymine.

After UV excitation, gas phase thymine returns to a ground state in 5 to 7 ps, showing multiple time constants. There is no consensus on the assignment of these processes, with a dispute between models claiming that thymine is trapped either in the first (S1) or in the second (S2) excited states. In the present study, a nonadiabatic dynamics simulation of thymine is performed on the basis of ADC(2) surfaces, to understand the role of dynamic electron correlation on the deactivation pathways. The results show that trapping in S2 is strongly reduced in comparison to previous simulations considering only non-dynamic electron correlation on CASSCF surfaces. The reason for the difference is traced back to the energetic cost for formation of a CO π bond in S2.

Localized operator partitioning method for electronic excitation energies in the time-dependent density functional formalism.

We extend the localized operator partitioning method (LOPM) [J. Nagesh, A. F. Izmaylov, and P. Brumer, J. Chem. Phys. 142, 084114 (2015)] to the time-dependent density functional theory framework to partition molecular electronic energies of excited states in a rigorous manner. A molecular fragment is defined as a collection of atoms using Becke's atomic partitioning. A numerically efficient scheme for evaluating the fragment excitation energy is derived employing a resolution of the identity to preserve standard one- and two-electron integrals in the final expressions. The utility of this partitioning approach is demonstrated by examining several excited states of two bichromophoric compounds: 9-((1- naphthyl)- methyl)- anthracene and 4-((2- naphthyl)- methyl)- benzaldehyde. The LOPM is found to provide nontrivial insights into the nature of electronic energy localization that is not accessible using a simple density difference analysis.

Fast Numerical Evaluation of Time-Derivative Nonadiabatic Couplings for Mixed Quantum-Classical Methods.

We have developed a numerical differentiation scheme that eliminates evaluation of overlap determinants in calculating the time-derivative nonadiabatic couplings (TDNACs). Evaluation of these determinants was the bottleneck in previous implementations of mixed quantum-classical methods using numerical differentiation of electronic wave functions in the Slater determinant representation. The central idea of our approach is, first, to reduce the analytic time derivatives of Slater determinants to time derivatives of molecular orbitals and then to apply a finite-difference formula. Benchmark calculations prove the efficiency of the proposed scheme showing impressive several-order-of-magnitude speedups of the TDNAC calculation step for midsize molecules.

An efficient implementation of the localized operator partitioning method for electronic energy transfer.

The localized operator partitioning method [Y. Khan and P. Brumer, J. Chem. Phys. 137, 194112 (2012)] rigorously defines the electronic energy on any subsystem within a molecule and gives a precise meaning to the subsystem ground and excited electronic energies, which is crucial for investigating electronic energy transfer from first principles. However, an efficient implementation of this approach has been hindered by complicated one- and two-electron integrals arising in its formulation. Using a resolution of the identity in the definition of partitioning, we reformulate the method in a computationally efficient manner that involves standard one- and two-electron integrals. We apply the developed algorithm to the 9-((1-naphthyl)-methyl)-anthracene (A1N) molecule by partitioning A1N into anthracenyl and CH2-naphthyl groups as subsystems and examine their electronic energies and populations for several excited states using configuration interaction singles method. The implemented approach shows a wide variety of different behaviors amongst the excited electronic states.

Simulation of Ã(2)A(1)←X̃(2)E laser excitation spectrum of CH3O and CD3O.

A theoretical calculation of the laser-induced fluorescence excitation spectrum from X̃(2)E→Ã(2)A1 is carried out for CH3O and CD3O using a transition dipole moment surface expanded up to second order. The vibronic form of these operators is obtained using symmetry arguments. The Ã(2)A1 vibrational levels are calculated using Van Vleck perturbation theory, and the latter is used to adjust harmonic constants of the potential to match experimental fundamentals. The CH3O fit force field is tested on CD3O. For both molecules the transition energies are well reproduced, but there are systematic differences between experimental and theoretical intensities. The origins of these differences are discussed.

Infrared spectra at a conical intersection: vibrations of methoxy.

The J = 0 infrared spectrum of methoxy is theoretically calculated for the ground X̃(2)E state using a quartic potential energy force field, and the quadratic dipole moment expansion is calculated ab initio at the CCSD(T) level of theory and cc-pVTZ basis. Writing these expansions with vibronic operators whose symmetry properties are defined with respect to C(3v) rotation greatly simplifies these calculations. With minor adjustments to the force field, excellent agreement with experiment is found for both the transition energies of CH(3)O and those of CD(3)O. The role of Jahn-Teller and Fermi coupling is illustrated by scaling these terms by a parameter δ that varies from 0 to 1. Plotting the eigenvalues as a function of δ yields a correlation diagram connecting the harmonic eigenvalues to those of the fully coupled problem. The spectrum for CH(3)O is determined using a combination of Davidson and Lanczos iteration schemes. The spectral features are found to be dominated by Jahn-Teller effects, but direct Fermi coupling and indirect potential couplings have important roles. The origin of the complexities in the CH stretch region are discussed.

Vibrational dynamics around the conical intersection: a study of methoxy vibrations on the X(2)E surface.

The large-amplitude vibrational dynamics of methoxy (CH(3)O), resulting from the conical intersection at the C(3v) geometry, are investigated using variational methods. A two-dimensional model Hamiltonian consisting of two vibrational degrees of freedom and the coupling between them is presented in a diabatic representation of the electronic degrees of freedom. The observed complex dynamics are understood in terms of the multiple timescales that arise as the initial wave packet passes through the conical intersection. This model Hamiltonian is extended to include a full-dimensional treatment of methoxy. A quartic potential is calculated using both single and multiple configuration methods. The vibronic eigenstates are calculated using a series of basis set contraction schemes taking a primitive basis of harmonic oscillator wave functions as the starting point. Our final results, including spin-orbit coupling, are compared to experiment.