Krisztina Varga

Ligand Induced Circular Dichroism and Circularly Polarized Luminescence in CdSe Quantum Dots

Chiral thiol capping ligands L- and D-cysteines induced modular chiroptical properties in achiral cadmium selenide quantum dots (CdSe QDs). Cys-CdSe prepared from achiral oleic acid capped CdSe by postsynthetic ligand exchange displayed size-dependent electronic circular dichroism (CD) and circularly polarized luminescence (CPL). Opposite CPL signals were measured for the CdSe QDs capped with D- and L-cysteine. The CD profile and CD anisotropy varied with size of CdSe nanocrystals with largest anisotropy observed for CdSe nanoparticles of 4.4 nm. Magic angle spinning solid state NMR (MAS ssNMR) experiments suggested bidentate interaction between cysteine and the surface of CdSe. Time Dependent Density Functional Theory (TDDFT) calculations verified that attachment of L- and D-cysteine to the surface of model (CdSe)13 nanoclusters induces measurable opposite CD signals for the exitonic band of the nanocluster. The origin of the induced chirality is consistent with the hybridization of highest occupied CdSe molecular orbitals with those of the chiral ligand.

Achiral CdSe quantum dots exhibit optical activity in the visible region upon post-synthetic ligand exchange with D- or L-cysteine

Semiconductor cadmium selenide (CdSe) quantum dots (QDs) exhibited mirror-image circular dichroism (CD) spectra in the visible region (350-570 nm) after replacing the trioctylphosphine oxide/oleic acid ligands on achiral nanocrystals with D- and L-cysteines. Chiroptical properties of cysteine-capped CdSe QDs depend on their size and can be fine-tuned by changing the radius of QDs.

Structure of the BamC two-domain protein obtained by Rosetta with a limited NMR data set

The CS-RDC-NOE Rosetta program was used to generate the solution structure of a 27-kDa fragment of the Escherichia coli BamC protein from a limited set of NMR data. The BamC protein is a component of the essential five-protein β-barrel assembly machine in E. coli. The first 100 residues in BamC were disordered in solution. The Rosetta calculations showed that BamC₁₀₁₋₃₄₄ forms two well-defined domains connected by an ~18-residue linker, where the relative orientation of the domains was not defined. Both domains adopt a helix-grip fold previously observed in the Bet v 1 superfamily. ¹⁵N relaxation data indicated a high degree of conformational flexibility for the linker connecting the N-terminal domain and the C-terminal domain in BamC. The results here show that CS-RDC-NOE Rosetta is robust and has a high tolerance for misassigned nuclear Overhauser effect restraints, greatly simplifying NMR structure determinations.

The Conformation of Bacteriorhodopsin Loops in Purple Membranes Resolved by Solid-State MAS NMR Spectroscopy†

Membrane proteins (and rhodopsin-like G-protein coupled receptors (GPCRs), in particular) are of significant biological and medical importance since they represent over 50% (GPCRs 25%) of current drug targets. However, the structure determination of membrane proteins is challenging: currently they account for < 1% of the unique protein structures deposited in the Protein Databank. X-ray crystallography has been used to make major contributions towards the structure determination of membrane proteins, but it suffers from the fact that the proteins are rarely crystallized in their native lipid environment or are unmodified, and exposed loop regions are often either dynamic and not visible, or involved in crystal contacts. NMR spectroscopic studies of membrane proteins in solution are generally also reliant on an artificial detergent environment and are also limited by protein size. Solid-state NMR (ssNMR) spectroscopy, in contrast, has the advantage that membrane proteins can be studied in a lipid environment. Although ssNMR does not suffer from the same intrinsic size limitation as solution NMR spectroscopy, spectral overlap is often severe for large proteins and hampers their study. However, magicangle-spinning (MAS) NMR spectroscopy, in particular, has been used to make substantial methodological advances in recent years, and the first membrane protein structures have now been determined using this technique. Herein we report on how solid-state MAS NMR spectroscopy can be used to complement X-ray crystallographic studies of a large seven transmembrane (7TM) helical protein by validating and redefining the loop structures. The structure of bacteriorhodopsin (bR) has been determined in a range of two(2D) and three-dimensional (3D) crystalline environments with the loops showing the highest degree of structural variation. Solid-state MAS NMR spectra of uniformly [C,N]-labeled bR in its native purple membrane have been used to assign the signals of the loop regions of the protein. Extraction of dihedral angle information from chemical shifts has allowed us to validate several loop conformations in the crystal structure and recalculate the structure where there are differences in conformation. Ab initio assignment of the resonances of the loop regions of bR was carried out using 2D DARR spectra (mixing times of 15 and 50 ms) and 3D NCACX (20 ms), 3D NCOCX (20 ms), 3D CANCO and 3D CAN(CO)CX (45 ms) spectra. Assignment of the loops is made possible by the fact that the loop resonances are generally well separated and amenable to assignment in contrast to many of the helical regions, where leucine and valine resonances, in particular, exhibit intractable degrees of spectral overlap. Figure 1 shows the assignment of the section Met68–Gly72 in the BC loop as an example; further 2D spectra and strip plots are provided in the Supporting Information (Figures S1–S3). In total, we have assigned roughly 55% of loop residues covering all loops, except for the CD loop, as well as several residues located in the helices (Figure 2, Table S2 in the Supporting Information, and BMRB Accession code 17361). Interestingly, residues from all loops (except those in the unassigned CD loop) are visible in our cross-polarization (CP)-based spectra; this is in contrast to the spectra of sensory rhodopsin II from Natronomonas pharaonis (NpSRII) where most loops were visible only in INEPT-based spectra, reflecting a higher degree of loop mobility in NpSRII. Our observations are more similar to those made in a recent study of proteorhodopsin in which only isolated residues were observed in INEPT-based spectra. A C,C INEPT-COSY spectrum of bR contains resonances with random-coil chemical shifts from amino acid types that are consistent with the Nand C-terminal tails (see Figures S4 and S5 in Supporting Information). Some chemical shifts for side chains in non-random-coil conformations are also found for residues Lys, Glu, Ala, and Ser, which may belong to the KAES motif in the EF loop. Sequential [*] Dr. V. A. Higman, Dr. P. J. Judge, Prof. A. Watts Department of Biochemistry, University of Oxford South Parks Road, Oxford, OX1 3QU (UK) E-mail: anthony.watts@bioch.ox.ac.uk Dr. K. Varga Department of Chemistry, University of Wyoming Laramie, WY 82071 (USA)

Caulobacter PopZ forms an intrinsically disordered hub in organizing bacterial cell poles

Significance Despite being the simplest organisms, bacteria have complex subcellular anatomies. How does such organization occur in openly diffusive cytoplasm? We find that the cell pole organizing protein PopZ facilitates network formation by binding directly to at least eight other proteins. The binding region in PopZ is intrinsically disordered, suggesting PopZ has a flexible structure that adopts a different interface for each partner protein. In this way, PopZ resembles eukaryotic hub proteins, such as p53 and BRCA1, which coordinate complex signaling networks. Rapid cycles of binding, unbinding, and rebinding within PopZ networks indicate that bacterial cell poles, similar to their eukaryotic counterparts, are highly dynamic structures. Despite their relative simplicity, bacteria have complex anatomy at the subcellular level. At the cell poles of Caulobacter crescentus, a 177-amino acid (aa) protein called PopZ self-assembles into 3D polymeric superstructures. Remarkably, we find that this assemblage interacts directly with at least eight different proteins, which are involved in cell cycle regulation and chromosome segregation. The binding determinants within PopZ include 24 aa at the N terminus, a 32-aa region near the C-terminal homo-oligomeric assembly domain, and portions of an intervening linker region. Together, the N-terminal 133 aa of PopZ are sufficient for interacting with all binding partners, even in the absence of homo-oligomeric assembly. Structural analysis of this region revealed that it is intrinsically disordered, similar to p53 and other hub proteins that organize complex signaling networks in eukaryotic cells. Through live-cell photobleaching, we find rapid binding kinetics between PopZ and its partners, suggesting many pole-localized proteins become concentrated at cell poles through rapid cycles of binding and unbinding within the PopZ scaffold. We conclude that some bacteria, similar to their eukaryotic counterparts, use intrinsically disordered hub proteins for network assembly and subcellular organization.

The effects of high concentrations of ionic liquid on GB1 protein structure and dynamics probed by high-resolution magic-angle-spinning NMR spectroscopy

Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentrations of ionic liquids, has been challenging. In the present work the 13C, 15N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid–protein interactions. Effect of an ionic liquid (1-butyl-3-methylimidazolium bromide, [C4-mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6–3.5 M, which corresponds to 10–60% v/v). Interactions between GB1 and [C4-mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in 15N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C4-mim]Br were assigned using 3D methods under HR-MAS conditions. Thus, HR-MAS NMR is a viable tool that could aid in elucidation of molecular mechanisms of ionic liquid–protein interactions.

CdSe Quantum Dots Functionalized with Chiral, Thiol-Free Carboxylic Acids: Unraveling Structural Requirements for Ligand-Induced Chirality

Functionalization of colloidal quantum dots (QDs) with chiral cysteine derivatives by phase-transfer ligand exchange proved to be a simple yet powerful method for the synthesis of chiral, optically active QDs regardless of their size and chemical composition. Here, we present induction of chirality in CdSe by thiol-free chiral carboxylic acid capping ligands (l- and d-malic and tartaric acids). Our circular dichroism (CD) and infrared experimental data showed how the presence of a chiral carboxylic acid capping ligand on the surface of CdSe QDs was necessary but not sufficient for the induction of optical activity in QDs. A chiral bis-carboxylic acid capping ligand needed to have three oxygen-donor groups during the phase-transfer ligand exchange to successfully induce chirality in CdSe. Intrinsic chirality of CdSe nanocrystals was not observed as evidenced by transmission electron microscopy and reverse phase-transfer ligand exchange with achiral 1-dodecanethiol. Density functional theory geometry optimizations and CD spectra simulations suggest an explanation for these observations. The tridentate binding via three oxygen-donor groups had an energetic preference for one of the two possible binding orientations on the QD (111) surface, leading to the CD signal. By contrast, bidentate binding was nearly equienergetic, leading to cancellation of approximately oppositely signed corresponding CD signals. The resulting induced CD of CdSe functionalized with chiral carboxylic acid capping ligands was the result of hybridization of the (achiral) QD and (chiral) ligand electronic states controlled by the ligands absolute configuration and the ligands geometrical arrangement on the QD surface.

Theoretical and experimental study of the antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability

In this work the physico-chemical properties of selected cryoprotectants (antifreeze protein TrxA-AFP752, trehalose and dimethyl sulfoxide) were correlated with their impact on the constitution of ice and influence on frozen/thawed cell viability. The freezing processes and states of investigated materials solutions were described and explained from a fundamental point of view using ab-initio modelling (molecular dynamics, DFT), Raman spectroscopy, Differential Scanning Calorimetry and X-Ray Diffraction. For the first time, in this work we correlated the microscopic view (modelling) with the description of the frozen solution states and put these results in the context of human skin fibroblast viability after freezing and thawing. DMSO and AFP had different impacts on their solutions freezing process but in both cases the ice crystallinity size was considerably reduced. DMSO and AFP treatment in different ways improved the viability of frozen/thawed cells.

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Changes in cryopreserved cell nuclei serve as indicators of processes during freezing and thawing

The mechanisms underlying cell protection from cryoinjury are not yet fully understood. Recent biological studies have addressed cryopreserved cell survival but have not correlated the cryoprotection effectiveness with the impact of cryoprotectants on the most important cell structure, the nucleus, and the freeze/thaw process. We identified changes of cell nuclei states caused by different types of cryoprotectants and associate them with alterations of the freeze/thaw process in cells. Namely, we investigated both higher-order chromatin structure and nuclear envelope integrity as possible markers of freezing and thawing processes. Moreover, we analyzed in detail the relationship between nuclear envelope integrity, chromatin condensation, freeze/thaw processes in cells, and cryopreservation efficiency for dimethyl sulfoxide, glycerol, trehalose, and antifreeze protein. Our interdisciplinary study reveals how changes in cell nuclei induced by cryoprotectants affect the ability of cells to withstand freezing and thawing and how nuclei changes correlate with processes during freezing and thawing. Our results contribute to the deeper fundamental understanding of the freezing processes, notably in the cell nucleus, which will expand the applications and lead to the rational design of cryoprotective materials and protocols.