Rapid Crystallization of l-Alanine on Engineered Surfaces by Use of Metal-Assisted and Microwave-Accelerated Evaporative Crystallization

This study demonstrates the application of metal-assisted and microwave-accelerated evaporative crystallization (MA-MAEC) technique to rapid crystallization of l-alanine on surface-engineered silver nanostructures. In this regard, silver island films (SIFs) were modified with hexamethylenediamine (HMA), 1-undecanethiol (UDET), and 11-mercaptoundecanoic acid (MUDA), which introduced -NH₂, -CH₃, and -COOH functional groups to SIFs, respectively. l-Alanine was crystallized on these engineered surfaces and blank SIFs at room temperature by the MA-MAEC technique. Significant improvements in crystal size, shape, and quality were observed on HMA-, MUDA- and UDET-modified SIFs at room temperature (crystallization time = 144, 40, and 147 min, respectively) as compared to those crystals grown on blank SIFs. By use of the MA-MAEC technique, the crystallization time of l-alanine on engineered surfaces was reduced to 17 s for microwave power level 10 (i.e., duty cycle 100%) and 7 min for microwave power level 1 (duty cycle 10%). Raman spectroscopy and powder X-ray diffraction (XRD) measurements showed that l-alanine crystals grown on engineered surfaces by the MA-MAEC technique had identical characteristic peaks to l-alanine crystals grown by traditional evaporative crystallization

Short Carboxylic Acid–Carboxylate Hydrogen Bonds Can Have Fully Localized Protons

Short hydrogen bonds (H-bonds) have been proposed to play key functional roles in several proteins. The location of the proton in short H-bonds is of central importance, as proton delocalization is a defining feature of low-barrier hydrogen bonds (LBHBs). Experimentally determining proton location in H-bonds is challenging. Here, bond length analysis of atomic (1.15–0.98 Å) resolution X-ray crystal structures of the human protein DJ-1 and its bacterial homologue, YajL, was used to determine the protonation states of H-bonded carboxylic acids. DJ-1 contains a buried, dimer-spanning 2.49 Å H-bond between Glu15 and Asp24 that satisfies standard donor–acceptor distance criteria for a LBHB. Bond length analysis indicates that the proton is localized on Asp24, excluding a LBHB at this location. However, similar analysis of the <i>Escherichia coli</i> homologue YajL shows both residues may be protonated at the H-bonded oxygen atoms, potentially consistent with a LBHB. A Protein Data Bank-wide screen identifies candidate carboxylic acid H-bonds in approximately 14% of proteins, which are typically short [⟨<i>d</i>_O–O⟩ = 2.542(2) Å]. Chemically similar H-bonds between hydroxylated residues (Ser/Thr/Tyr) and carboxylates show a trend of lengthening O–O distance with increasing H-bond donor p<i>K</i>_a. This trend suggests that conventional electronic effects provide an adequate explanation for short, charge-assisted carboxylic acid–carboxylate H-bonds in proteins, without the need to invoke LBHBs in general. This study demonstrates that bond length analysis of atomic resolution X-ray crystal structures provides a useful experimental test of certain candidate LBHBs

Additional file 3: Figure S3. of A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone

Two dimers of E6a bound hNQO1 structure showing the loop 230–236 interacting with the active site of neighboring dimer. The FAD molecules are shown in stick representation in each active site. The E6a molecule is shown in ball-and-stick representation. (PNG 2799 kb

Additional file 4: Figure S4. of A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone

The chemical structures of other known inhibitors of NQO1. (PNG 79 kb

Cysteine p<i>K</i>_a Depression by a Protonated Glutamic Acid in Human DJ-1

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined p<i>K</i>_a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p<i>K</i>_a of 5.4 ± 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine p<i>K</i>_a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the p<i>K</i>_a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its p<i>K</i>_a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid−cysteine interactions, which we propose are particularly common in the DJ-1 superfamily

Additional file 1: Figure S1. of A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone

The chemical structure of E6a (3-bromo-3′-hydroxy-2,2′-binaphthalenyl-1,4,1′,4′-tetraone). (PNG 40 kb

Compounds studied in this work.

1, 7-nitroindole-2-carboxylic acid or CRT0044876; 2, 5-fluoroindole-2-carboxylic acid; 3, 5-nitroindole-2-carboxylic acid; 4, 6-bromoindole-2-carboxylic acid; 5, myricetin; 6, AR03 or MLS000552981 of the NIH MLSMR library; 7, APE1 Inhibitor III, MLS000419194, or N-(3-(benzo[d]- thiazol-2-yl)-6-isopropyl-4,5,6,7-tetrahydrothieno[2,3-c]- pyridin-2-yl)acetamide. For compounds 1–4, the 2-carboxyl is in the deprotonated form that likely predominates at pH 7.5.</p

Structural Basis for Excision of 5‑Formylcytosine by Thymine DNA Glycosylase

Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epigenetic regulation. Performing a critical step in a pathway for active DNA demethylation, TDG removes 5-formylcytosine and 5-carboxylcytosine, oxidized derivatives of 5-methylcytosine that are generated by TET (ten–eleven translocation) enzymes. We determined a crystal structure of TDG bound to DNA with a noncleavable (2′-fluoroarabino) analogue of 5-formyldeoxycytidine flipped into its active site, revealing how it recognizes and hydrolytically excises fC. Together with previous structural and biochemical findings, the results illustrate how TDG employs an adaptable active site to excise a broad variety of nucleobases from DNA

S4 File -

(PDB)</p

APE1 chemical shift perturbations induced by MgCl₂ (1.0 mM).

(a) 15N-TROSY spectra for APE1 (0.10 mM) in the absence (black) or presence (red) of MgCl2 (1.0 mM). Spectra were also collected for APE1 with lower MgCl2 concentrations (0.063, 0.125, 0.25, 0.50. 0.75 mM). Two residues near the Mg2+-binding site (69, 308) exhibit peaks for apo APE1 but not APE1 with MgCl2 (≥0.063 mM). (b) CSPs (Δδ) induced by MgCl2 (1.0 mM) versus amino acid residue. Labels with stars mark residues for which a peak is not seen in spectra of APE1 with 1.0 mM MgCl2; for these residues, Δδ values were calculated using spectra for APE1 with the highest [MgCl2] for which that peak is observed (*, 0.063 mM; **, 0.125 mM; ***, 0.25 mM; ****, 0.50 mM; *****, 0.75 mM). Residues exhibiting Δδ ≥ 0.015 ppm are labeled. (TIF)</p