Initial Excited State Dynamics of Lumichrome upon Ultraviolet Excitation.

Lumichrome (LC) is the major photodegradation product of biologically important flavin cofactors. Since LC serves as a structural comparison with the flavins; understanding excited states of LC is fundamentally important to establish a connection with photophysics of different flavins, such as lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Herein, we deduce the initial excited state structural dynamics of LC using UV resonance Raman (UVRR) intensity analysis.

Developing a Coarse-Grained Model for Bacterial Cell Walls: Evaluating Mechanical Properties and Free Energy Barriers.

The bacterial cell envelope of Gram-negative bacteria is a complex biological barrier with multiple layers consisting of the inner membrane, periplasm of peptidoglycan, and the outer membrane with lipopolysaccharides (LPS). With rising antimicrobial resistance there is increasing interest in understanding interactions of small molecules with the cell membrane to aid in the development of novel drug molecules. Hence suitable representations of the bacterial membrane are required to carry out meaningful molecular dynamics simulations.

Assessing Barriers for Antimicrobial Penetration in Complex Asymmetric Bacterial Membranes: A Case Study with Thymol.

The bacterial cell envelope is a complex multilayered structure evolved to protect bacteria in hostile environments. An understanding of the molecular basis for the interaction and transport of antibacterial therapeutics with the bacterial cell envelope will enable the development of drug molecules to combat bacterial infections and suppress the emergence of drug-resistant strains. Here we report the successful creation of an in vitro supported lipid bilayer (SLB) platform of the outer membrane (OM) of , an archetypical Gram-negative bacterium, containing the full smooth lipopolysaccharide (S-LPS) architecture of the membrane.

Kinetics of Melanin Polymerization during Enzymatic and Nonenzymatic Oxidation.

Melanin is an abundant biopigment in the animal kingdom, but its structure remains poorly understood. This is a substantial impediment to understanding the mechanistic origin of its observed functions. Proposed models of melanin structure include aggregates of both linear and macrocyclic units and noncovalently held monomers.

Structural basis for substrate discrimination by repair enzyme, AlkB.

AlkB, a repair enzyme of the dioxygenase family, catalyses the removal of mutagenic methylated nucleotides from the genome. Known for substrate promiscuity, AlkB's catalytic mechanism and conformational changes accompanying substrate binding have been extensively dissected. However, the structural parameters of various substrates governing their recognition by AlkB still remain elusive.

Ultrafast structural dynamics of photoexcited adenine.

We report deep UV initiated excited state dynamics of the canonical nucleobase adenine (Ade) through Resonance Raman (RR) intensity analysis. RR spectra of Ade at excitation wavelengths throughout the B absorption band in the 210-230 nm wavelength range are measured and subsequently converted to scattering cross-sections. The time-dependent wave packet (TDWP) formalism has been employed for self-consistent simulations of the resulting wavelength dependent Raman excitation profiles (REP) and absorption spectrum of Ade.

Ultrafast Nuclear Dynamics of Photoexcited Guanosine-5'-Monophosphate in Three Singlet States.

We report measurement of resonance Raman (RR) spectra of guanosine-5'-monophosphate (GMP), a DNA nucleotide at excitation wavelengths throughout its ππ* absorption band (B) in the 210-230 nm range. From these data, we constructed wavelength-dependent Raman intensity excitation profiles (REPs) for all observed modes. These profiles and the absorption spectrum were then modeled using self-consistent simulations based on the time-dependent wave packet propagation formalism.

Mechanism of Discrimination of 8-Oxoguanosine versus Guanosine by Escherichia coli Fpg.

The mutagenic 8-oxoguanosine monophosphate, the predominant product of DNA oxidation, is excised by formamidopyrimidine glycosylase (Fpg) in bacteria. The mechanism of recognition of 8-oxodG, which differs subtly from its normal counterpart, guanosine monophosphate (dG), by Escherichia coli Fpg remains elusive due to the lack of structural data of E. coli Fpg bound to 8-oxodG.

Sub-50 fs excited state dynamics of 6-chloroguanine upon deep ultraviolet excitation.

The photophysical properties of natural nucleobases and their respective nucleotides are ascribed to the sub-picosecond lifetime of their first singlet states in the UV-B region (260-350 nm). Electronic transitions of the ππ* type, which are stronger than those in the UV-B region, lie at the red edge of the UV-C range (100-260 nm) in all isolated nucleobases. The lowest energetic excited states in the UV-B region of nucleobases have been investigated using a plethora of experimental and theoretical methods in gas and solution phases.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction.

Solution structure of ligands involved in purine salvage pathway.

Analogues of intermediates involved in the purine salvage pathway can be exploited as potential drug molecules against enzymes of protozoan parasites. To develop such analogues we need knowledge of the solution structures, predominant tautomer at physiological pH and protonation-state of the corresponding natural ligand. In this regard, we have employed ultraviolet resonance Raman spectroscopy (UVRR) in combination with density functional theory (DFT) to study the solution structures of two relatively unexplored intermediates, 6-phosphoryl IMP (6-pIMP) and succinyl adenosine-5'-monophosphate (sAMP), of purine salvage pathway.

Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine-Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides.

Plasmodium falciparum (Pf) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9-deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs.

Solution structures of purine base analogues 9-deazaguanine and 9-deazahypoxanthine.

Deaza analogues of nucleobases are potential drugs against infectious diseases caused by parasites. A caveat is that apart from binding their target parasite enzymes, they also bind and inhibit enzymes of the host. In order to design derivatives of deaza analogues which specifically bind target enzymes, knowledge of their molecular structure, protonation state, and predominant tautomers at physiological conditions is essential.

Formation of a CH-π contact in the core of native barstar during folding.

An important part of the protein folding process is the consolidation of the protein core through the formation of specific, directional contacts after the initial hydrophobic collapse. Here, we simultaneously monitor formation of core contacts and assembly of secondary structure through salt-induced folding by using resonance Raman spectroscopy. Unfolded barstar at pH 12 was refolded by gradual addition of sodium sulfate salt.

Resonance Raman spectroscopic measurements delineate the structural changes that occur during tau fibril formation.

The aggregation of the microtubule-associated protein, tau, into amyloid fibrils is a hallmark of neurodegenerative diseases such as the tauopathies and Alzheimer's disease. Since monomeric tau is an intrinsically disordered protein and the polymeric fibrils possess an ordered cross-β core, the aggregation process is known to involve substantial conformational conversion besides growth. The aggregation mechanism of tau in the presence of inducers such as heparin, deciphered using probes such as thioflavin T/S fluorescence, light scattering, and electron microscopy assays, has been shown to conform to ligand-induced nucleation-dependent polymerization.

Mode-specific reorganization energies and ultrafast solvation dynamics of Tryptophan from Raman line-shape analysis.

Tryptophan is widely used as an intrinsic fluorophore for studies of protein structure and dynamics. Its fluorescence is known to have two decay components with lifetimes of 0.5 and 3.

Solution structures of purine base analogues 6-chloroguanine, 8-azaguanine and allopurinol.

Analogues of purine bases are highly relevant in the biological context and have been implicated as drug molecules for therapy against a number of diseases. Additionally, these molecules have been implicated to have a role in the prebiotic RNA world. However, experimental data on the structures of these molecules in aqueous solution is lacking.

Methylation stabilizes the imino tautomer of dAMP and amino tautomer of dCMP in solution.

Alkylating agents cause methylation of adenosine and cytidine in DNA to generate 1-methyladenosine and 3-methylcytidine. These modified nucleosides can serve as regulators of cells or can act as agents of mutagenesis depending on the context and the partner enzymes. Solution structures and the chemical interactions with enzymes that lead to their recognition are of inherent interest.

Hypoxanthine guanine phosphoribosyltransferase distorts the purine ring of nucleotide substrates and perturbs the pKa of bound xanthosine monophosphate.

Enzymatic efficiency and structural discrimination of substrates from nonsubstrate analogues are attributed to the precise assembly of binding pockets. Many enzymes have the additional remarkable ability to recognize several substrates. These apparently paradoxical attributes are ascribed to the structural plasticity of proteins.

Vibrational markers of structural distortion in adenine nucleobases upon DNA damage.

Oxidation is one of the common causes of chemical damage of DNA. Among the oxidized nucleobases in DNA, 8-oxoadenine (8-oxoA) and 4,6-diamino-5-formamidoadenine (FaPyA) are two of the most commonly found lesions. Relatively little information has been published so far on these lesions compared to the more mutagenic modified purines like 8-oxoguanine.

Structures, ionization equilibria, and tautomerism of 6-oxopurines in solution.

6-Oxopurine and its analogues form an important class of biological molecules that include nucleobases and their precursors and are substrates of a wide range of enzymes. Solution structures of purines have been debated in the literature because of the many possible tautomers and protonation states in which they can exist in solution. Substitutions on the pyrimidine and imidazole rings alter tautomerization and protonation equilibria, and as a consequence, the solution compositions and structures of closely related analogues can be significantly different.

Solution structure of the DNA damage lesion 8-oxoguanosine from ultraviolet resonance Raman spectroscopy.

UV radiation and reactive byproducts of cellular metabolism are constant threats to genomic stability. A frequent consequence is the oxidation of DNA nucleobases, especially guanine to 8-oxoguanosine. This highly mutagenic lesion can form base pairs with other nucleobases, does not significantly distort the DNA structure, and remains unnoticed by DNA polymerases.

Heme displacement mechanism of CooA activation: mutational and Raman spectroscopic evidence.

The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F.

Dynamics of carbon monoxide binding to cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric.

Dynamics of carbon monoxide binding to CooA.

CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes.