Evaluation of π-π interactions in proteins using Trp analogs:From protein labeling to quantitating the energies involved

Zijketens van aromatische aminozuren zijn vaak betrokken bij kation-π en π-π interacties. Hoewel deze interacties belangrijk zijn voor de stabiliteit en functie van eiwitten, is slechts weinig fundamentele kennis beschikbaar over de sterkte van deze interacties. Een elegante methode om hierin inzicht te krijgen is via het inbouwen van aromatische aminozuren met één tot vier fluoratomen in de zijketen. Echter, de bestaande methoden om deze eiwitten te produceren geven extreem lage opbrengsten. In dit proefschrift wordt een Lactococcus lactis tryptofaan auxotroof expressiesysteem beschreven om deze eiwitten in milligram hoeveelheden en met een hoge inbouwefficiëntie (≥ 95%) te produceren. Gebruik werd gemaakt van een synthetisch groeimedium dat uit slechts 24 componenten bestond, terwijl de meest gebruikte synthetische media voor L. lactis veelal 50 componenten bevatten. Met dit expressiesysteem werd een reeks tryptofaan analogen ingebouwd in het LmrR eiwit. LmrR is een translatie repressor eiwit dat dimeren vormt en aromatische liganden kan binden tussen twee tryptofaan residuen in het dimeergrensvlak. Gevonden werd dat, naarmate meer fluor atomen aanwezig waren in de tryptofaan zijketen, des te lager werd de affiniteit van LmrR voor zijn liganden. Dit is een sterke aanwijzing dat π-π interacties een belangrijke rol spelen om het ligand te binden. Hoge-resolutiestructuren van LmrR met gebonden ligand bevestigen dat de inbouw van fluoratomen in een aromatisch aminozuur geen grote structurele veranderingen induceert. Het hier gepresenteerde L. lactis tryptofaan auxotroof expressiesysteem maakt het nu dus mogelijk om voldoende eiwit te maken om π-π interacties te bestuderen met diverse biofysische methoden

On the Adversarial Robustness of Vision Transformers

Following the success in advancing natural language processing and understanding, transformers are expected to bring revolutionary changes to computer vision. This work provides the first and comprehensive study on the robustness of vision transformers (ViTs) against adversarial perturbations. Tested on various white-box and transfer attack settings, we find that ViTs possess better adversarial robustness when compared with convolutional neural networks (CNNs). This observation also holds for certified robustness. We summarize the following main observations contributing to the improved robustness of ViTs: 1) Features learned by ViTs contain less low-level information and are more generalizable, which contributes to superior robustness against adversarial perturbations. 2) Introducing convolutional or tokens-to-token blocks for learning low-level features in ViTs can improve classification accuracy but at the cost of adversarial robustness. 3) Increasing the proportion of transformers in the model structure (when the model consists of both transformer and CNN blocks) leads to better robustness. But for a pure transformer model, simply increasing the size or adding layers cannot guarantee a similar effect. 4) Pre-training on larger datasets does not significantly improve adversarial robustness though it is critical for training ViTs. 5) Adversarial training is also applicable to ViT for training robust models. Furthermore, feature visualization and frequency analysis are conducted for explanation. The results show that ViTs are less sensitive to high-frequency perturbations than CNNs and there is a high correlation between how well the model learns low-level features and its robustness against different frequency-based perturbations

Ag nanoparticles/PPV composite nanofibers with high and sensitive opto-electronic response

The novel Ag nanoparticles/poly(p-phenylene vinylene) [PPV] composite nanofibers were prepared by electrospinning. The transmission electron microscope image shows that the average diameter of composite fibers is about 500 nm and Ag nanoparticles are uniformly dispersed in the PPV matrix with an average diameter of about 25 nm. The Fourier transform infrared spectra suggest that there could be a coordination effect to a certain extent between the Ag atom and the π system of PPV, which is significantly favorable for the dissociation of photoexcitons and the charge transfer at the interface between the Ag nanoparticle and the PPV. The Au top electrode device of the single Ag/PPV composite nanofiber exhibits high and sensitive opto-electronic responses. Under light illumination of 5.76 mW/cm2 and voltage of 20 V, the photocurrent is over three times larger than the dark current under same voltage, which indicates that this kind of composite fiber is an excellent opto-electronic nanomaterial

The Role of Tryptophan in π Interactions in Proteins:An Experimental Approach

In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π-π, cation-π, and CH-π bonds. These interactions are often crucial for protein structure and protein-ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96' in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π-π interactions for drug binding. Presteady state kinetic data of the LmrR-drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein-drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15-20% to the total riboflavin-RibU binding energy

Incorporation of tryptophan analogues into the lantibiotic nisin

Lantibiotics are posttranslationally modified peptides with efficient inhibitory activity against various Gram-positive bacteria. In addition to the original modifications, incorporation of non-canonical amino acids can render new properties and functions to lantibiotics. Nisin is the most studied lantibiotic and contains no tryptophan residues. In this study, a system was constructed to incorporate tryptophan analogues into nisin, which included the modification machinery (NisBTC) and the overexpression of tryptophanyl-tRNA synthetase (TrpRS). Tryptophan and three different tryptophan analogues (5-fluoroTrp (5FW), 5-hydroxyTrp (5HW) and 5-methylTrp (5MeW)) were successfully incorporated at four different positions of nisin (I1W, I4W, M17W and V32W). The incorporation efficiency of tryptophan analogues into mutants I1W, M17W and V32W was over 97 %, while the mutant I4W showed relatively low incorporation efficiency (69-93 %). The variants with 5FW showed relatively higher production yield, while 5MeW-containing variants showed the lowest yield. The dehydration efficiency of serines or threonines was affected by the tryptophan mutants of I4W and V32W. The affinity of the peptides for the cation-ion exchange and reverse phase chromatography columns was significantly reduced when 5HW was incorporated. The antimicrobial activity of IIW and its 5FW analogue both decreased two times compared to that of nisin, while that of its 5HW analogue decreased four times. The 5FW analogue of I4W also showed two times decreased activity than nisin. However, the mutant M17W and its 5HW analogue both showed 32 times reduced activity relative to that of nisin

CO2 saturated brine injected into fractured shale: An X-ray micro-tomography in-situ analysis at reservoir conditions

Fracture morphology and permeability are key factors in enhanced gas recovery (EOR) and Carbon Geo-storage (CCS) in shale gas reservoirs as they determine production and injection rates. However, the exact effect of CO2-saturated (live) brine on shale fracture morphology, and how the permeability changes during live brine injection and exposure is only poorly understood. We thus imaged fractured shale samples before and after live brine injection in-situ at high resolution in 3D via X-ray micro-computed tomography. Clearly, the fractures’ aperture and connectivity increased after live brine injection