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.     

Stress fiber dynamics as probed by antibodies against myosin

The dynamics of microfilament bundles (stress fibers) in tissue culture cells were studied by microinjecting an affinity-purified polyclonal antibody against chicken gizzard myosin. This antibody cross-reacted exclusively with the light chains of nonmuscle myosin and should therefore bind to the head portion of myosin molecules. When injected in high concentrations (13-26 mg/ml), it disrupted stress fibers in a high proportion (60-80%) of rat and chicken embryo fibroblasts, as well as in PtK2 cells. Myosin was found collected in large aggregates probably comprising protein: antibody precipitates, while actin and alpha-actinin were not localized in any defined structures in stress fiber depleted cells. Fibroblasts rounded up, probably because of lack of tension-generating microfilament bundles. After several hours, stress fibers were seen to regrow again in the afflicted cells, even when myosin precipitates and excess antibody were still present. The extent of stress fiber disruption and the time point of their reappearance were dependent on the concentration of the injected antibody.     

VASP governs actin dynamics by modulating filament anchoring

Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell.     

Computer-assisted tracking of actin filament motility.

In vitro motility assays, in which fluorescently labeled actin filaments are propelled by myosin molecules adhered to a glass coverslip, require that actin filament velocity be determined. We have developed a computer-assisted filament tracking system that reduced the analysis time, minimized investigator bias, and provided greater accuracy in locating actin filaments in video images. The tracking routine successfully tracked filaments under experimental conditions where filament density, size, and extent of photobleaching varied dramatically. Videotaped images of actin filament motility were digitized and processed to enhance filament image contrast relative to background. Once processed, filament images were cross correlated between frames and a filament path was determined. The changes in filament centroid or center position between video frames were then used to calculate filament velocity. The tracking routine performance was evaluated and the sources of noise that contributed to errors in velocity were identified and quantified. Errors originated in algorithms for filament centroid determination and in the choice of sampling interval between video frames. With knowledge of these error sources, the investigator can maximize the accuracy of the velocity calculation through access to user-definable computer program parameters.     

Excited-state dynamics of bacteriorhodopsin probed by broadband femtosecond fluorescence spectroscopy

The impact of varying excitation densities (approximately 0.3 to approximately 40 photons per molecule) on the ultrafast fluorescence dynamics of bacteriorhodopsin has been studied in a wide spectral range (630-900 nm). For low excitation densities, the fluorescence dynamics can be approximated biexponentially with time constants of <0.15 and approximately 0.45 ps. The spectrum associated with the fastest time constant peaks at 650 nm, while the 0.45 ps component is most prominent at 750 nm. Superimposed on these kinetics is a shift of the fluorescence maximum with time (dynamic Stokes shift). Higher excitation densities alter the time constants and their amplitudes. These changes are assigned to multi-photon absorptions.     

Catalytically requisite conformational dynamics in the mRNA-capping enzyme probed by targeted molecular dynamics

The addition of a N7-methyl guanosine cap to the 5' end of nascent mRNA is carried out by the mRNA-capping enzyme, a two-domain protein that is a member of the nucleotidyltransferase superfamily. The mRNA-capping enzyme is composed of a catalytic nucleotidyltransferase domain and a noncatalytic oligonucleotide/oligosaccharide binding (OB) domain. Large-scale domain motion triggered by substrate binding mediates catalytically requisite conformational rearrangement of the GTP substrate prior to the chemical step. In this study, we employ targeted molecular dynamics (TMD) on the PBCV-1 capping enzyme to probe the global domain dynamics and internal dynamics of conserved residues during the conformational transformation from the open to the closed state. Analysis of the resulting trajectories along with structural and sequence homology to other members of the superfamily allows us to suggest a conserved mechanism of conformational rearrangements spanning all mRNA-capping enzymes and all ATP-dependent DNA ligases. Our results suggest that the OB domain moves quasi-statically toward the nucleotidyltransferase domain, pivoting about a short linker region. The approach of the OB domain brings a conserved RxDK sequence, an element of conserved motif VI, within proximity of the triphosphate of GTP, destabilizing the unreactive conformation and thereby allowing thermal fluctuations to partition the substrate toward the catalytically competent state.     

Sugar transport across lactose permease probed by steered molecular dynamics

Escherichia coli lactose permease (LacY) transports sugar across the inner membrane of the bacterium using the proton motive force to accumulate sugar in the cytosol. We have probed lactose conduction across LacY using steered molecular dynamics, permitting us to follow molecular and energetic details of lactose interaction with the lumen of LacY during its permeation. Lactose induces a widening of the narrowest parts of the channel during permeation, the widening being largest within the periplasmic half-channel. During permeation, the water-filled lumen of LacY only partially hydrates lactose, forcing it to interact with channel lining residues. Lactose forms a multitude of direct sugar-channel hydrogen bonds, predominantly with residues of the flexible N-domain, which is known to contribute a major part of LacY's affinity for lactose. In the periplasmic half-channel lactose predominantly interacts with hydrophobic channel lining residues, whereas in the cytoplasmic half-channel key protein-substrate interactions are mediated by ionic residues. A major energy barrier against transport is found within a tight segment of the periplasmic half-channel where sugar hydration is minimal and protein-sugar interaction maximal. Upon unbinding from the binding pocket, lactose undergoes a rotation to permeate either half-channel with its long axis aligned parallel to the channel axis. The results hint at the possibility of a transport mechanism, in which lactose permeates LacY through a narrow periplasmic half-channel and a wide cytoplasmic half-channel, the opening of which is controlled by changes in protonation states of key protein side groups.     

Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths, with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm(-1), which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.     

Structural dynamics and folding of β-lactoglobulin probed by heteronuclear NMR

Bovine β-lactoglobulin (βLG) has been one of the most extensively studied proteins in the history of protein science mainly because its abundance in cow's milk makes it readily available to researchers. However, compared to other textbook proteins, progress in the study of βLG has been slow because of obstacles such as a low reversibility from denaturation linked with thiol–disulfide exchange or monomer–dimer equilibrium preventing a detailed NMR analysis. Recently, the expression of various types of recombinant βLGs combined with heteronuclear NMR analysis has significantly improved understanding of the physico-chemical properties of βLG. In this review, we address several topics including pH-dependent structural dynamics, ligand binding, and the complex folding mechanism with non-native intermediates. These unique properties might be brought about by conformational frustration of the βLG structure, partly attributed to the relatively large molecular size of βLG. We expect studies with βLG to continue to reveal various important findings, difficult to obtain with small globular proteins, leading to a more comprehensive understanding of the conformation, dynamics and folding of proteins.     

Misfolding pathways of the prion protein probed by molecular dynamics simulations

Although the cellular monomeric form of the benign prion protein is now well characterized, a model for the monomer of the misfolded conformation (PrP(Sc)) remains elusive. PrP(Sc) quickly aggregates into highly insoluble fibrils making experimental structural characterization very difficult. The tendency to aggregation of PrP(Sc) in aqueous solution implies that the monomer fold must be hydrophobic. Here, by using molecular dynamics simulations, we have studied the cellular mouse prion protein and its D178N pathogenic mutant immersed in a hydrophobic environment (solution of CCl4), to reveal conformational changes and/or local structural weaknesses of the prion protein fold in unfavorable structural and thermodynamic conditions. Simulations in water have been also performed. Although observing in general a rather limited conformation activity in the nanosecond timescale, we have detected a significant weakening of the antiparallel beta-sheet of the D178N mutant in CCl4 and to a less extent in water. No weakening is observed for the native prion protein. The increase of beta-structure in the monomer, recently claimed as evidence for misfolding to PrP(Sc), has been also observed in this study irrespective of the thermodynamic or structural conditions, showing that this behavior is very likely an intrinsic characteristic of the prion protein fold.     

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles.     

Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy.

The photoinduced reaction cycle of bacteriorhodopsin (BR) has been studied by means of a recently developed picosecond infrared spectroscopic method at ambient temperature. BR - K difference spectra between 1560 and 1700 cm-1 have been recorded at delay times from 100 ps to 14 ns. The spectrum remains unchanged during this period. The negative difference OD band at 1660 cm-1 indicates the peptide backbone responds within 50 ps. A survey in the region of carboxylic side chain absorption around 1740 cm-1 reveals that perturbations of those groups, present in low-temperature FTIR spectra, are not observable within 10 ns, suggesting a slow conformational change.     

Yeast kinetochore microtubule dynamics analyzed by high-resolution three-dimensional microscopy

We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.     

Resident-invader dynamics of similar strategies in fluctuating environments

Journal of Mathematical Biology - We study resident-invader dynamics in fluctuating environments when the invader and the resident have close but distinct strategies. First we focus on a class of...     

Steady state dynamics of intermediate filament networks

We have conducted experiments to examine the dynamic exchange between subunit and polymer of vimentin intermediate filaments (IF) at steady state through the use of xrhodamine-labeled vimentin in fluorescence recovery after photobleaching (FRAP) analysis. The xrhodamine-vimentin incorporated into the endogenous vimentin IF network after microinjection into fibroblasts and could be visualized with a cooled charge-coupled device (CCD) camera and digital imaging fluorescence microscopy. Bar shaped regions were bleached in the fluorescent IF network using a beam from an argon ion laser and the cells were monitored at various times after bleaching to assess recovery of fluorescence in the bleached zones. We determined that bleached vimentin fibers can recover their fluorescence over relatively short time periods. Vimentin fibers in living cells also can exhibit significant movements, but the recovery of fluorescence was not dependent upon movement of fibers. Fluorescence recovery within individual fibers did not exhibit any marked polarity and was most consistent with a steady state exchange of vimentin subunits along the lengths of IF.     

Structure and dynamics of the actin filament

The structures of filamentous Mg-ATP-actin (F actin) in the presence and absence of KCl have been mapped with hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry (MS) analysis revealed 52 reactive side-chain sites from 27 distinct peptides within actin. The reactivities of these probe sites with *OH in the F-actin states are compared with those of Mg-ATP-G-actin (monomers) analyzed previously [Guan, J.-Q. et al. (2003) Biochemistry 42, 11992-12000]. Filament-dependent protection within subdomains 2, 3, and 4 and at the C terminus is consistent with longitudinal contacts of monomers within the filament helical structure as predicted by the Holmes model. In the absence of KCl, the extent of filament-dependent protection rarely reached 3-fold, consistent with a highly dynamic filament characterized by relatively weak interactions between actin protomers. However, in the presence of KCl, the extents of protection are significantly increased, consistent with a well-ordered, more tightly packed filament structure. Filament-dependent enhancements of reactivity not predicted by the Holmes model are seen for a peptide that overlaps the "hydrophobic plug" (H-plug) region and for a peptide that forms contacts with the polyphosphate moiety of the bound nucleotide. Overall, these data are both consistent with and complementary to a recent deuterium-exchange MS study of filamentous actin [Chik, J. K., and Schriemer, D.C. (2003) J. Mol. Biol. 334, 373-385], which also did not detect any burial of the H plug upon formation of filaments.     

The open state of human topoisomerase I as probed by molecular dynamics simulation

The open state of human topoisomerase I has been probed by molecular dynamics simulation, starting from the coordinates of the closed structure of the protein complexed with DNA, after elimination of the 22-bp DNA duplex oligonucleotide. A repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum, after which an unperturbed simulation has been carried out for 10ns. The simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains, which however move as a coherent unit, fully maintaining their secondary and tertiary structures. Despite movements between the domains as large as 80–90Å, the catalytic pentad remains preassembled, the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7Å. Electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center, in a conformation perfectly suited to interact with the negatively charged supercoiled DNA substrate.