Long String Scattering in c = 1 String Theory

We study the scattering of long strings in c = 1 string theory, both in the worldsheet description and in the non-singlet sector of the dual matrix quantum mechanics. From the worldsheet perspective, the scattering amplitudes of long strings are obtained from a decoupling limit of open strings amplitudes on FZZT branes, which we compute by integrating Virasoro conformal blocks along with structure constants of boundary Liouville theory. In particular, we study the tree level amplitudes of (1) a long string decaying by emitting a closed string, and (2) the scattering of a pair of long strings. We show that they are indeed well defined as limits of open string amplitudes, and that our results are in striking numerical agreement with computations in the adjoint and bi-adjoint sectors of the dual matrix model (based on proposals of Maldacena and solutions due to Fidkowski), thereby providing strong evidence of the duality.Comment: 42 pages, 18 figure

The c=1 String Theory S-Matrix Revisited

We revisit the perturbative S-matrix of c=1 string theory from the worldsheet perspective. We clarify the origin of the leg pole factors, the non-analyticity of the string amplitudes, and the validity as well as limitations of earlier computations based on resonance momenta. We compute the tree level 4-point amplitude and the genus one 2-point reflection amplitude by numerically integrating Virasoro conformal blocks with DOZZ structure constants on the sphere and on the torus, with sufficiently generic complex Liouville momenta, and find agreement with known answers from the c=1 matrix model.Comment: 32 pages, 7 figures; footnote and references added, typos correcte

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently

Delineating Heme-Mediated versus Direct Protein Oxidation in Peroxidase-Activated Cytochrome

Oxidation of key residues in cytochrome c (cyt c) by chloramine T (CT) converts the protein from an electron transporter to a peroxidase. This peroxidase-activated state represents an important model system for exploring the early steps of apoptosis. CT-induced transformations include oxidation of the distal heme ligand Met80 (MetO, +16 Da) and carbonylation (LysCHO, -1 Da) in the range of Lys53/55/72/73. Remarkably, the 15 remaining Lys residues in cyt c are not susceptible to carbonylation. The cause of this unusual selectivity is unknown. Here we applied top-down mass spectrometry (MS) to examine whether CT-induced oxidation is catalyzed by heme. To this end, we compared the behavior of cyt c with (holo-cyt c) and without heme (apoSS-cyt c). CT caused MetO formation at Met80 for both holo- and apoSS-cyt c, implying that this transformation can proceed independently of heme. The aldehyde-specific label Girard\u27s reagent T (GRT) reacted with oxidized holo-cyt c, consistent with the presence of several LysCHO. In contrast, oxidized apo-cyt c did not react with GRT, revealing that LysCHO forms only in the presence of heme. The heme dependence of LysCHO formation was further confirmed using microperoxidase-11 (MP11). CT exposure of apoSS-cyt c in the presence of MP11 caused extensive nonselective LysCHO formation. Our results imply that the selectivity of LysCHO formation at Lys53/55/72/73 in holo-cyt c is caused by the spatial proximity of these sites to the reactive (distal) heme face. Overall, this work highlights the utility of top-down MS for unravelling complex oxidative modifications

Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome

Many aspects of protein function rely on conformational fluctuations. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) provides a window into these dynamics. Despite the widespread use of HDX-MS, it remains unclear whether this technique provides a truly comprehensive view of protein dynamics. HDX is mediated by H-bond-opening/closing events, implying that HDX methods provide an H-bond-centric view. This raises the question if there could be fluctuations that leave the H-bond network unaffected, thereby rendering them undetectable by HDX-MS. We explore this issue in experiments on cytochrom

Cytochrome c as a Peroxidase: Activation of the Precatalytic Native State by H

In addition to serving as respiratory electron shuttle, ferri-cytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by H2O2. This peroxidase function plays a key role during apoptosis. Typical peroxidases have a five-coordinate heme with a vacant distal coordination site that permits the iron center to interact with H2O2. In contrast, native cyt c is six-coordinate, as the distal coordination site is occupied by Met80. It thus seems counterintuitive that native cyt c would exhibit peroxidase activity. The current work scrutinizes the origin of this structure-function mismatch. Cyt c-catalyzed peroxidase reactions show an initial lag phase that is consistent with the in situ conversion of a precatalyst to an active peroxidase. Using mass spectrometry, we demonstrate the occurrence of cyt c self-oxidation in the presence of H2O2. The newly generated oxidized proteoforms are shown to possess significantly enhanced peroxidase activity. H2O2-induced modifications commence with oxidation of Tyr67, followed by permanent displacement of Met80 from the heme iron. The actual peroxidase activation step corresponds to subsequent side chain carbonylation, likely at Lys72/73. The Tyr67-oxidized/carbonylated protein has a vacant distal ligation site, and it represents the true peroxidase-active structure of cyt c. Subsequent self-oxidation eventually causes deactivation. It appears that this is the first report that identifies H2O2-induced covalent modifications as an essential component for the peroxidase activity of native cyt c

The S-Matrix of 2D Type 0B String Theory Part 2: D-Instanton Effects

We study the effect of D-instantons on closed string scattering amplitudes in the two-dimensional type 0B string theory from the worldsheet perspective. We find that the contribution from a pair of D-instanton and anti-D-instanton to the closed string reflection amplitude, with a suitable contour prescription for the integration over the D-instanton moduli space, agrees with the corresponding leading non-perturbative corrections in the proposed dual matrix quantum mechanics. This analysis is further extended to thermal observables defined at finite temperature. The infrared divergence in charged processes is understood through the measure factor for charged D-instantons, and can be treated with spacetime dimensional regularization.Comment: 30 pages, 3 figures, 1 tabl

Effects of Oxidative Modifications on the Structure and Non-Canonical Functions of Cytochrome c Studied by Mass Spectrometry

The peroxidase activity of the mitochondrial protein cytochrome c (cyt c) plays a critical role in triggering programmed cell death, or apoptosis. However, the native structure of cyt c should render this activity impossible due to the lack of open iron coordination sites at its heme cofactor. Despite its key biological importance, the molecular mechanisms underlying this structure-function mismatch remain enigmatic. The work detailed in this dissertation fills this knowledge gap by using mass spectrometry (MS) to decipher the central role that protein oxidative modifications and their associated structural changes play in activating the peroxidase function of cyt c. Chapter 2 uses a suite of MS-based experiments to identify and characterize oxidative modifications in cyt c caused by the oxidant and canonical peroxidase substrate, H2O2. In doing so, we unravel the critical role that these in situ structural changes play in triggering the peroxidase activity of the protein via alteration of the coordination environment. Serendipitously, we also discover that certain functionally important oxidative modifications, particularly on Lys, can elude detection when using conventional bottom-up MS approaches. However, by applying top-down MS we could successfully detect these modifications. Chapter 3 re-examines a popular and purportedly well-characterized model system for peroxidase-activated cyt c: cyt c treated with chloramine-T. By combining top-down MS with sample fractionation techniques, we uncover that this model system is in fact comprised of a broad ensemble of structurally and functionally distinct species. These species can be differentiated by the extent of oxidation at key Lys residues, which previously went undetected. Chapter 4 expands on the previous chapters by probing the causal factors underpinning the production of oxidative modification products at Lys and other residues. We discover that Lys oxidation is catalyzed by the endogenous heme cofactor, while other transformations (e.g. Met oxidation) proceed via direct interaction with the oxidant. Chapter 5 utilizes oxidized cyt c as a model system to test the compatibility of protein stability measurements in the gas phase to their counterparts in solution. Unlike many other protein systems, we discover that oxidized cyt c shows opposing stability trends in solution and in the gas phase