Susanta K. Sarkar

An improved method for two-dimensional heteronuclear relayed-coherence-transfer NMR spectroscopy

Recently, a variety of schemes for obtaining 2D heteronuclear RELAY spectra have been proposed (I-6). These methods can be quite powerful for structure determination and spectral assignment. However, a recent analysis (7) has shown that in many cases the sensitivity of heteronuclear relayed-magnetization-transfer spectroscopy is considerably lower than for heteronuclear chemical-shift correlation (8-10). Especially in compounds with saturated aliphatic ring systems, such as steroids and alkaloids, the sensitivity of the heteronuclear RELAY experiment is often very low. Moreover, in all heteronuclear RELAY experiments proposed so far the ‘H-‘H multiplet components of the (‘H-13C) RELAY multiplet are in antiphase relative to one another (analogous to the antiphase nature of multiplet components in a COSY spectrum (II)) and the ‘H-13C RELAY multiplet is 90” out of phase relative to nonrelayed signal in the 2D spectrum. No pure-absorption 2D spectra can therefore be recorded with the existing RELAY experiments. We propose a new pulse scheme for heteronuclear RELAY spectroscopy that partly overcomes the problems mentioned above. In the new scheme, net magnetization transfer among protons is achieved via homonuclear Hartmann-Hahn-type cross-polarization. This type of homonuclear magnetization transfer was first discovered (12) as an artifact in homonuclear transverse NOE spectroscopy (13) and is closely related to the magnetization-transfer mechanism in the TOCSY experiment (14). We have recently demonstrated that this type of homonuclear cross-polarization is very powerful for the assignment of complex proton spectra (15). The possibility of obtaining net magnetization transfer in coupled ‘H spin systems makes this approach very suitable for heteronuclear RELAY spectroscopy. The pulse scheme of the new method is sketched in Fig. 1. An alternating spinlock field along the fx axis of the proton rotating frame provides the crucial mechanism for homonuclear Hartmann-Hahn-type cross-polarization. Consider first, for reasons of simplicity, a rf field of nominal strength, v = yH2/27r, that is continuously aligned along the x axis of the proton. rotating frame. Two protons, A and B, with offset frequencies AA and AB, experience effective rf field strengths, VA and VB . Provided that v s AA, AB, VA and VB are to a good approximation given by

Silica encapsulation of fluorescent nanodiamonds for colloidal stability and facile surface functionalization.

Fluorescent nanodiamonds (FNDs) emit in the near-IR and do not photobleach or photoblink. These properties make FNDs better suited for numerous imaging applications compared with commonly used fluorescence agents such as organic dyes and quantum dots. However, nanodiamonds do not form stable suspensions in aqueous buffer, are prone to aggregation, and are difficult to functionalize. Here we present a method for encapsulating nanodiamonds with silica using an innovative liposome-based encapsulation process that renders the particle surface biocompatible, stable, and readily functionalized through routine linking chemistries. Furthermore, the method selects for a desired particle size and produces a monodisperse agent. We attached biotin to the silica-coated FNDs and tracked the three-dimensional motion of a biotinylated FND tethered by a single DNA molecule with high spatial and temporal resolution.

Direct measurement of DNA bending by type IIA topoisomerases: implications for non-equilibrium topology simplification

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.

Single-Molecule Tracking of Collagenase on Native Type I Collagen Fibrils Reveals Degradation Mechanism

BACKGROUND Collagen, the most abundant human protein, is the principal component of the extracellular matrix and plays important roles in maintaining tissue and organ integrity. Highly resistant to proteolysis, fibrillar collagen is degraded by specific matrix metalloproteases (MMPs). Degradation of fibrillar collagen underlies processes including tissue remodeling, wound healing, and cancer metastasis. However, the mechanism of native collagen fibril degradation remains poorly understood. RESULTS Here we present the results of high-resolution tracking of individual MMPs degrading type I collagen fibrils. MMP1 exhibits cleavage-dependent biased and hindered diffusion but spends 90% ± 3% of the time in one of at least two distinct pause states. One class of exponentially distributed pauses (class I pauses) occurs randomly along the fibril, whereas a second class of pauses (class II pauses) exhibits multistep escape kinetics and occurs periodically at intervals of 1.3 ± 0.2 μm and 1.5 ± 0.2 μm along the fibril. After these class II pauses, MMP1 moved faster and farther in one direction along the fibril, indicative of biased motion associated with cleavage. Simulations indicate that 5% ± 2% of the class II pauses result in the initiation of processive collagen degradation, which continues for bursts of 15 ± 4 consecutive cleavage events. CONCLUSIONS These findings provide a mechanistic paradigm for type I collagen degradation by MMP1 and establish a general approach to investigate MMP-fibrillar collagen interactions. More generally, this work demonstrates the fundamental role of enzyme-substrate interactions including binding and motion in determining the activity of an enzyme on an extended substrate.

Wide-field in vivo background free imaging by selective magnetic modulation of nanodiamond fluorescence.

The sensitivity and resolution of fluorescence-based imaging in vivo is often limited by autofluorescence and other background noise. To overcome these limitations, we have developed a wide-field background-free imaging technique based on magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are bright, photo-stable, biocompatible nanoparticles that are promising probes for a wide range of in vitro and in vivo imaging applications. Our readily applied background-free imaging technique improves the signal-to-background ratio for in vivo imaging up to 100-fold. This technique has the potential to significantly improve and extend fluorescent nanodiamond imaging capabilities on diverse fluorescence imaging platforms.

Elimination of refocusing pulses in NMR experiments

A large number of one- and two-dimensional NMR experiments employ 180” refocusing pulses with the main purpose of obtaining absorption-mode spectra. Imperfections in the flip angle of the refocusing pulse will generally lead to degradation of the sensitivity of the experiment and may give rise to unwanted resonances in 2D spectra. Those problems can sometimes be significantly alleviated by proper use of the composite pulse concept (Z-4). However, we will demonstrate that those problems can be completely avoided by omitting the refocusing pulses altogether.

Tunable optical delay via carrier induced exciton dephasing in semiconductor quantum wells.

We report the experimental realization of a tunable optical delay by exploiting unique incoherent nonlinear optical processes in semiconductors. The tunable optical delay takes advantage of the strong Coulomb interactions between excitons and free carriers and uses optical injection of free carriers to broaden and bleach an exciton absorption resonance. Fractional delay exceeding 200% has been obtained for an 8 ps optical pulse propagating near the heavy-hole excitonic transition in a GaAs quantum well structure. Tunable optical delay based on optical injection of free carriers avoids strong absorption of the pump beam and is also robust against variations in the frequency of the pump beam.

Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling

Significance Collagen fibrils resemble nanoscale cables that self-assemble and constitute the most prevalent protein structure in the body. Our experiments reveal unanticipated defects that form along collagen fibrils. These defects are the initiation sites of collagenase activity and represent a strain-sensitive mechanism for regulating tissue remodeling. The emergence of defects, their spatial periodicity, and fluctuations are quantitatively accounted for with a buckling model in which defects spontaneously form, repulsively interact, and self-heal. Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal–strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments.

Optimization of heteronuclear relayed coherence-transfer spectroscopy

Abstract The effectiveness of the heteronuclear RELAY experiment is analyzed for a number of different spin systems. It is found that the sensitivity of the measurement strongly depends on the duration of the mixing period and on a proper choice for the sampling time in the t 1 dimension. Good agreement is found between theoretical evaluations and experimental results, obtained for samples of propanol and cyclosporin A.

Inducing electron spin coherence in GaAs quantum well waveguides: spin coherence without spin precession

Electron spin coherence is induced via light-hole transitions in a quantum well waveguide without a DC magnetic field. The spin coherence is detected through effects of quantum interference in spectral domain nonlinear optical response.