Dinesh K. Sukumaran

Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment

A suite of reduced-dimensionality 13C,15N,1H-triple-resonance NMR experiments is presented for rapid and complete protein resonance assignment. Even when using short measurement times, these experiments allow one to retain the high spectral resolution required for efficient automated analysis. “Sampling limited” and “sensitivity limited” data collection regimes are defined, respectively, depending on whether the sampling of the indirect dimensions or the sensitivity of a multidimensional NMR experiments per se determines the minimally required measurement time. We show that reduced-dimensionality NMR spectroscopy is a powerful approach to avoid the “sampling limited regime”—i.e., a standard set of ten experiments proposed here allows one to effectively adapt minimal measurement times to sensitivity requirements. This is of particular interest in view of the greatly increased sensitivity of NMR spectrometers equipped with cryogenic probes. As a step toward fully automated analysis, the program autoassign has been extended to provide sequential backbone and 13Cβ resonance assignments from these reduced-dimensionality NMR data.

STRUCTURE OF HUMAN SALIVARY HISTATIN 5 IN AQUEOUS AND NONAQUEOUS SOLUTIONS

The solution structure of human salivary histatin 5 (D-S-H-A-K-R-H-H-G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) was examined in water (pH 3.8) and dimethyl sulfoxide solutions using 500 MHz homo- and heteronuclear two-dimensional (2D) nmr. The resonance assignment of peptide backbone and side-chain protons was accomplished by 2D total correlated spectroscopy and nuclear Overhauser effect (NOE) spectroscopy. The high JNH-C alpha H values (> or = 7.4 Hz), absence of any characteristic NH-NH (i, i + 1) or C alpha H-C beta H (i, i + 3) NOE connectivities, high d delta/dT values (> or = 0.004 ppm K-1) and the fast 1H/2H amide exchange suggest that histatin 5 molecules remain unstructured in aqueous solution at pH 3.8. In contrast, histatin 5 prefers largely alpha-helical conformation in dimethyl sulfoxide solution as evident from the JNH-C alpha H values (< or = 6.4 Hz), slow 1H/2H exchange, low d delta/dT values (< or = 0.003 ppm K-1) observed for amide resonances of residues 6-24, and the characteristic NH-NH (i, i + 1) and C alpha H-C beta H (i, i + 3) NOE connectivities. All backbone amide 15N-1H connectivities fall within 6 ppm on the 15N scale in the 2D heteronuclear single quantum correlated spectrum, and the restrained structure calculations using DIANA suggest the prevalence of alpha-helical conformations stabilized by 19 (5-->1) intramolecular backbone amide hydrogen bonds in polar aprotic medium such as dimethyl sulfoxide. The interside-chain hydrogen bonding and salt-bridge type interactions that normally stabilize the helical structure of linear peptides in aqueous solutions are not observed. Histatin 5, unlike other naturally occurring antimicrobial polypeptides such as magainins, defensins, and tachyplesins, does not adopt amphiphilic structure, precluding its insertion into microbial membranes and formation of ion channels across membranes. Electrostatic (ionic type) and hydrogen bonding interactions of the positively charged and polar residues with the head groups of microbial membranes or with a membrane-bound receptor could be the initial step involved in the mechanism of antimicrobial activity of histatins.

INTERACTION OF MELITTIN WITH LIPID MEMBRANES

Interaction of melittin with lipid membranes was studied systematically with respect to its adsorption onto membranes, its effect on membrane leakage and fusion, and micellization at various melittin/lipid ratios. It was found that melittin has a strong affinity for adsorption onto lipid membranes. The analysis of the measured electrophoretic mobilities by use of a Gouy-Chapman double layer theory, shows that melittin is adsorbed onto the phosphatidylserine membrane several times more than the phosphatidylcholine membrane. However, it was observed that the phosphatidylcholine membrane is more susceptible to membrane leakage, vesicle fusion and micellization at a lower level of melittin adsorbed than the phosphatidylserine membrane. For small unilamellar phosphatidylcholine vesicles in 0.1 M NaCl, membrane leakage started at melittin to lipid ratio of 1:2000, a large increase in the rate of membrane leakage occurred at a ratio of about 1:500 or higher, membrane fusion occurred at a ratio of 1:200, and membrane micellization at a ratio of 1:10. On the other hand, for small unilamellar phosphatidylserine vesicles, the respective concentrations of melittin to result in membrane leakage, vesicle fusion, and membrane micellization were several times higher. Surface pressure measurements of lipid monolayers showed that the increase in surface pressure of the phosphatidylcholine monolayer due to the presence of melittin in the subphase solution was greater than that for the phosphatidylserine monolayer at any melittin concentration in the subphase solution. These experimental results indicate that melittin tends to be adsorbed on the surface of the negatively charged phosphatidylserine membrane due to the electrostatic binding so that the melittin molecule can stay out more on the surface of the membrane, while melittin appears to be adsorbed more into the hydrophobic membrane core for the electrically neutral phosphatidylcholine membrane.

Diagnosis of early stage ovarian cancer by 1H NMR metabonomics of serum explored by use of a microflow NMR probe.

We show that (1)H NMR based metabonomicsof serum allows the diagnosis of early stage I/II epithelial ovarian cancer (EOC) required for successful treatment. Because patient specimens are highly precious, we conducted an exploratory study using a microflow probe requiring only 20 μL of serum. By use of logistic regression on principal components (PCs) of the NMR profiles, we built a 4-variable model for early stage EOC prediction (training set: 69 EOC specimens, 84 healthy controls; test set: 40 EOC, 44 controls) with operating characteristics estimated for the test set at 80% specificity [95% confidence interval (CI): 65-90%], 63% sensitivity (95% CI: 46-77%), and an area under the Receiver Operator Characteristic Curve (AUC) of 0.796. Independent validation (50 EOC, 50 controls) of the model yielded 95% specificity (95% CI: 86-99.5%), 68% sensitivity (95% CI: 53-80%) and an AUC of 0.949. A test on cancer type specificity showed that women diseased with renal cell carcinoma were not incorrectly diagnosed with EOC, indicating that metabonomics bears significant potential for cancer type-specific diagnosis. Our model can potentially be applied for women at high risk for EOC, and our study promises to contribute to developing a screening protocol for the general population.

Ascorbate protects endothelial barrier function during septic insult: Role of protein phosphatase type 2A

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2A inhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-gamma). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier.

Highly selective synthesis of the ring-B reduced chlorins by ferric chloride-mediated oxidation of bacteriochlorins: effects of the fused imide vs isocyclic ring on photophysical and electrochemical properties.

The oxidation of bacteriopyropheophorbide with ferric chloride hexahydrate or its anhydrous form produced the ring-D oxidized (ring-B reduced) chlorin in >95% yield. Replacing the five-member isocyclic ring in bacteriopyropheophorbide- a with a fused six-member N-butylimide ring system made no difference in regioselective oxidation, and the corresponding ring-B reduced chlorin was isolated in almost quantitative yield. When the oxidant was replaced by 2,3-dichloro-5,6-dicyano-p-benzoquinone, which is frequently used at the oxidizing stage of the porphyrin synthesis, the ring-B oxidized (ring-D reduced) chlorins were obtained. With both ring-B reduced and ring-D reduced chlorins in hand, their photophysical and electrochemical properties were examined and compared for the first time. The ring-B reduced chlorine 20, with a fused six-member N-butylimide ring, exhibits the most red-shifted absorption band (at lambda(max) = 746 nm), the lowest fluorescence quantum yield (4.5%), and the largest quantum yield of singlet oxygen formation (67%) among the reduced ring-B and ring-D chlorins investigated in this study. Measurements of the one-electron oxidation and reduction potentials show that compound 20 is also the easiest to oxidize among the examined compounds and the third easiest to reduce. In addition, the 1.62 eV HOMO-LUMO gap of 20 is the smallest of the examined compounds, and this agrees with values calculated using the DFT method. Spectroelectrochemical measurements afforded UV-visible absorption spectra for both the radical cations and radical anions of the examined chlorins. The ring-B reduced compound 20, with a fused six-member N-butylimide ring, is regarded as the most promising candidate in this study for photodynamic therapy because it has the longest wavelength absorption and the largest quantum yield of singlet oxygen formation among the compounds investigated.

Synthesis, microbicidal activity, and solution structure of the dodecapeptide from bovine neutrophils

The dodecapepetide sequence R-L-C-R-I-V-V-I-R-V-C-R with a disulfide bridge between the cysteine residues found in bovine neutrophils was synthesized by solid-phase procedures. Its antimicrobial activity against oral microorganisms such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus gordonii was examined, and its structural features were examined by CD and determined by two-dimensional (2D) nmr. The strains P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are found to be highly sensitive to this peptide at 2-2.5 microM concentrations, suggesting that the dodecapeptide is a potent antibiotic for oral pathogens. The weak negative n-sigma* band observed at approximately 265-270 nm in the CD spectra of this peptide provides evidence for the presence of a disulfide bridge. The negative n-pi* band at approximately 200 nm and the positive pi-pi* band at 185 nm suggest a folded structure for this peptide. The negative n-pi* shifts from 200 to 206 nm with an increase in intensity in dipalmitoylphosphotidylcholine vesicles, suggesting that the peptide might associate to form higher order aggregates in lipid medium. The assignment of backbone and side-chain proton resonances has been accomplished by the combined analysis of 2D total correlated and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and (1)H-(2)H exchange effect on amide NH resonances indicate the involvement of amide NH groups of Cys3, Ile5, Ile8, Val10, and Arg12 in intramolecular hydrogen bonding. The coupling constant (J(NH-C(alpha)H)) values, the set of medium-, short-, and long-range nuclear Overhauser effects, and the results of restrained structure calculation using the distance geometry algorithm for nmr applications provide evidence for a folded, loop-like structure with a type I (III) beta-turn involving Ile5, Val6, Val7, and Ile8, and two antiparallel beta-strands involving the N-terminal Arg1, Leu2, Cys3, and Val4 and the C-terminal Arg9, Val10, Cys11, and Arg12 residues. The structure of the dodecapeptide mimics the amphiphilic structure of large 30-35 residue defensins and the peptide appears to exhibit similar antimicrobial potency.

Discrete Stacking of Aromatic Oligoamide Macrocycles

Unlike the precise structural control typical of closed assemblies, curbing the stacking of disc- and ring-shaped molecules is quite challenging. Here we report the discrete stacking of rigid aromatic oligoamide macrocycles 1. With increasing concentration, the aggregation of 1 quickly plateaus, forming a discrete oligomer, as suggested by 1D (1)H, 2D nuclear Overhauser effect, and diffusion-ordered NMR spectroscopy. Quantum-chemical calculations indicate that the tetramer of 1 is the most stable among oligomeric stacks. X-ray crystallography revealed a tetrameric stack containing identical molecules adopting two different conformations. With a defined length and an inner pore capable of accommodating distinctly different guests, the tetramers of 1 densely pack into 2D layers. Besides being a rare system of conformation-regulated supramolecular oligomerization, the discrete stacks of 1, along with their higher-order assemblies, may offer new nanotechnological applications.

Standard operating procedure for metabonomics studies of blood serum and plasma samples using a 1H-NMR micro-flow probe†

A standard operating procedure (SOP) is presented for high‐throughput metabonomics studies using a 600 MHz NMR spectrometer equipped with a micro‐flow probe that is connected to an auto‐sampler. The procedure is designed to minimize random and systematic variation of NMR data collection. In addition, a protocol is described to assess the quality of the data acquired by a given NMR spectroscopist to ensure that (i) all researchers involved in the NMR data acquisition of a metabonomics research program perform equally and (ii) operator‐associated variation of NMR data collection is statistically not relevant for the interpretation of results obtained from multivariate data analyses. Copyright

NMR structure of the hypothetical protein NMA1147 from Neisseria meningitidis reveals a distinct 5-helix bundle.

Gaohua Liu, Dinesh K. Sukumaran, Duanxiang Xu, Yiwen Chiang, Thomas Acton, Sharon Goldsmith-Fischman, Barry Honig, Gaetano T. Montelione, and Thomas Szyperski* Department of Chemistry, University at Buffalo, the State University of New York, Buffalo, New York Center of Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York Northeast Structural Genomics Consortium