Christopher J. Petzold

PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor Sulfolipid-1

Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. A diacylated intermediate in SL-1 biosynthesis, SL1278, has been shown to activate the adaptive immune response in human patients. Although several proteins involved in SL-1 biosynthesis have been identified, the enzymes that acylate the T2S core to form SL1278 and SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL1278 and are essential for SL-1 biosynthesis. In vitro, recombinant PapA2 converts T2S to 2′-palmitoyl T2S, and PapA1 further elaborates this newly identified SL-1 intermediate to an analog of SL1278. Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Finally, the ΔpapA2 and ΔpapA1 mutants were screened for virulence defects in a mouse model of infection. The loss of SL-1 (and SL1278) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection.

Laser-induced acoustic desorption/chemical ionization in Fourier-transform ion cyclotron resonance mass spectrometry

Abstract Laser-induced acoustic desorption (LIAD) of neutral molecules coupled with electron and chemical ionization was examined as an analysis method for nonvolatile organic and biomolecules in Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry. LIAD involved the production of a high amplitude acoustic wave by laser ablation of a copper or titanium foil from the opposite side of where the sample was deposited. The experiment was carried out with a simple probe designed for transmission mode laser desorption. Large amounts of neutral molecules were desorbed this way, but ions were not detected. The desorbed neutral molecules were ionized by 70 eV electron ionization or by reactions with reagent ions that were generated, isolated, and trapped in the ICR cell. Strong, reproducible signals were obtained in these experiments. The applicability of the method was demonstrated for a wide variety of molecules, including an organic salt, steroids, sugars, oligopeptides, nucleic acid bases, nucleosides, and synthetic polymers. For example, the tetrapeptide val-ala-ala-phe was volatilized by LIAD and ionized by a proton transfer reaction. The ions were stored in the FT-ICR cell for up to 30 s before detection. The base peak in the spectrum obtained corresponds to the protonated peptide, which indicates that the neutral peptide was desorbed intact. In contrast, thermal desorption of this peptide leads to substantial degradation. Based on this and other results obtained, LIAD combined with postdesorption ionization in an FT-ICR shows promise as a practical method for the analysis of thermally labile, nonvolatile molecules. A special advantage of this approach is that it allows better control of the ionization step (through selection of reagent ions) and a broader choice of ionization modes (e.g. group transfer, addition/elimination) than laser desorption/ionization methods, such as matrix-assisted laser desorption/ionization (MALDI). A disadvantage is lower sensitivity.

Gas-phase reactions of charged phenyl radicals with neutral biomolecules evaporated by laser-induced acoustic desorption.

A generally applicable method for the study of phenyl radicals’ reactions with neutral biomolecules in the gas phase is demonstrated. Neutral biomolecules were evaporated into a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR) by means of laser-induced acoustic desorption (LIAD) and subsequently reacted with trapped charged phenyl radicals. The structural integrity of the evaporated alanylalanine molecules was verified by reaction with dichlorophosphenium ions. Examination of the reactions of charged phenyl radicals with alanylalanine and thymidine evaporated via LIAD revealed hydrogen atom abstraction for both alanylalanine and thymidine as well as an addition/elimination product for the reaction with thymidine. These reactions are consistent with the results obtained by others in solution. Further, a previously unstudied reaction of the nucleotide of thymine (T1) with charged phenyl radical was found to yield analogous products as the reaction with thymidine.

Laser desorption in transmission geometry inside a Fourier-transform ion cyclotron resonance mass spectrometer

We report here the first application of laser desorption (LD) in transmission geometry (backside irradiation of the sample through a transparent support) inside a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR). A probe-mounted fiber optic assembly was used to simplify the implementation of this LD technique. This setup requires little or no instrument modifications, has minimum maintenance requirements, and is relatively inexpensive to build. The performance of the probe was tested by determining the molecular weight of a commercial polystyrene standard from its matrix-assisted laser desorption/ionization (MALDI) spectrum. The measured average molecular weight is comparable to that obtained for the same sample by MALDI in the conventional top-illumination arrangement (reflection geometry) and by the manufacturer of the sample by gel permeation chromatography. The average velocities measured for ions evaporated by transmission mode LD of several neat samples are about half the velocity of those obtained by using the reflection geometry. Therefore, transmission mode irradiation of the sample holds promise to desorb ions that are easier to trap in an ICR cell. An oscillating capillary nebulizer was adapted for the deposition of analytes to improve sampling reproducibility.

Examination of barriered and barrierless hydrogen atom abstraction reactions by organic radical cations: the cytosine radical cation

Abstract The radical cations of the nucleobases cytosine and adenine are easily generated in a dual cell Fourier transform ion cyclotron resonance mass spectrometer by using the technique of laser-induced acoustic desorption coupled with electron ionization. The cytosine radical cation is found to undergo facile hydrogen atom abstraction reactions with a variety of neutral reagents even when barrierless, exothermic electron transfer reactions are expected to dominate. In contrast, the adenine radical cation undergoes hydrogen atom abstraction only slowly in most of the cases studied. The ionic curve-crossing model is used to rationalize these results. Some of the thermodynamic and molecular properties of a radical cation that are favorable for efficient hydrogen atom abstraction are described in this article. Based on this analysis, dimethyl sulfoxide radical cation was predicted to abstract hydrogen atoms efficiently. Indeed, it was found to abstract hydrogen atoms at rates similar to cytosine radical cation. The transition from barriered to barrierless hydrogen atom abstraction that occurs as the ionization energy of the neutral hydrogen atom donor is lowered was used to “bracket” the recombination energy of cytosine radical cation at 8.9 ± 0.2 eV.

The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface

Achieving fast electron transfer between a material and protein is a long-standing challenge confronting applications in bioelectronics, bioelectrocatalysis, and optobioelectronics. Interestingly, naturally occurring extracellular electron transfer proteins bind to and reduce metal oxides fast enough to enable cell growth, and thus could offer insight into solving this coupling problem. While structures of several extracellular electron transfer proteins are known, an understanding of how these proteins bind to their metal oxide substrates has remained elusive because this abiotic-biotic interface is inaccessible to traditional structural methods. Here, we use advanced footprinting techniques to investigate binding between the Shewanella oneidensis MR-1 extracellular electron transfer protein MtrF and one of its substrates, α-Fe2O3 nanoparticles, at the molecular level. We find that MtrF binds α-Fe2O3 specifically, but not tightly. Nanoparticle binding does not induce significant conformational changes in MtrF, but instead protects specific residues on the face of MtrF likely to be involved in electron transfer. Surprisingly, these residues are separated in primary sequence, but cluster into a small 3D putative binding site. This binding site is located near a local pocket of positive charge that is complementary to the negatively charged α-Fe2O3 surface, and mutational analysis indicates that electrostatic interactions in this 3D pocket modulate MtrF-nanoparticle binding. Strikingly, these results show that binding of MtrF to α-Fe2O3 follows a strategy to connect proteins to materials that resembles the binding between donor-acceptor electron transfer proteins. Thus, by developing a new methodology to probe protein-nanoparticle binding at the molecular level, this work reveals one of natures strategies for achieving fast, efficient electron transfer between proteins and materials.