Stefan Wennmalm

The fluctuating enzyme: a single molecule approach

Abstract The excess of structural degrees of freedom in a protein enzyme opens questions about the conformational homogeneity. We studied single horseradish peroxidase enzyme turnovers by fluorescence spectroscopy. Application of a two-state dynamic model to the measured data shows exponential product dissociation kinetics, but a large distribution of rates for the enzyme to form the enzyme-product complex. The experiments show that in addition to the peroxidative cycle thermodynamic fluctuation phenomena on a wide range of time scales affect enzyme activity.

Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

INVERSE-FLUORESCENCE CORRELATION SPECTROSCOPY

An alternative version of fluorescence correlation spectroscopy is presented, where the signal from a medium surrounding the particles of interest is analyzed, as opposed to a signal from the particles themselves. This allows for analysis of unlabeled particles and potentially of biomolecules. Here, the concept together with principal experiments on polystyrene beads of 100, 200, 400, and 800 nm diameter in an aqueous solution of alexa 488-fluorophores are presented. The use of photo detectors allowing higher photon fluxes, or of reduced detection volumes, should enable analysis of significantly smaller particles or even biomolecules.

Inverse-Fluorescence Cross-Correlation Spectroscopy

Inverse-fluorescence correlation spectroscopy (iFCS) was recently introduced as an alternative version of FCS that does not require labeling of the analyzed particles or biomolecules. In iFCS, the signal from a medium surrounding the particles is analyzed, as opposed to a signal from the studied particles themselves. As unlabeled particles diffuse through the detection volume, they displace a fraction of the fluorescent medium, causing transient dips in the detected signal which give information about the mobility and concentration of the analyzed particles. Here inverse-fluorescence cross-correlation spectroscopy (iFCCS) is introduced as an extension of iFCS. In iFCCS, labeled particles/biomolecules are analyzed and their fluorescence signal is cross-correlated with the signal from the surrounding medium. When labeled particles are analyzed, a direct estimate of the volume of the particles is obtained or, alternatively, an estimate of the size of the detection volume. Another possibility is to analyze the interaction of small, labeled molecules with unlabeled particles, resulting in cross-correlation as an indication of binding, even though only one binding partner is labeled. This also enables accurate estimation of the degree of labeling, since the amounts of labeled and unlabeled particles are estimated independently.

UV-Fluorescence correlation spectroscopy of 2-aminopurine.

Abstract We have built a fluorescence correlation spectroscopy (FCS) microscope for ultraviolet excitation (280 300 nm) and emission. With UV excitation the fluorescence of natural fluorophores such as the modified nucleotide 2-aminopurine can be analyzed. The sensitivity of a natural fluorophore toward conformational changes can reveal dynamics in biomolecules. UVFCS is well suited for detection of intensity fluctuations related to such conformational dynamics. Here we show UVFCS measured on pQuarterphenyl and on 2-aminopurine (2-AP). The triplet state rate constants and the excitation cross section for 2- AP were estimated to k[23]=1 x 10[6] s[-1], k[31]=3 x 10[5] s[-1], and σ=2 x 10[-17] cm[2].

NON-ERGODIC BEHAVIOUR IN CONFORMATIONAL TRANSITIONS OF SINGLE DNA MOLECULES

Abstract Conformational fluctuations in single nucleic acid molecules have recently been observed through excited state lifetime measurements. Immobilisation of the sample molecule has also enabled direct observation of the fluorescence intensity fluctuations generated as the molecule switches between two conformations. As a probe for conformational fluctuations we use tetramethylrhodamine linked to a 217-bp DNA oligonucleotide. The measurements on this and similar systems reveal the existence of a distribution of reaction rates between the conformations. Here we report 37 detected single-molecule conformational fluctuations collected with the same immobilisation method as described earlier. Within the time window of observation the reaction rates differ between the molecules, but stay constant within a single molecule. The distribution of the relaxation rates between the molecules correspond to the distribution seen in a bulk measurement on a similar system. We therefore conclude that within the observation time window the single DNA molecules behave in a non-ergodic way.

Heterogeneity in single DNA conformational fluctuations

Abstract Non-exponential behaviour in bulk measurements of two-state molecular reactions can have different origins. The probability density for the time which one of two states is occupied during different observation interval times can be used to reveal system characteristics if single molecules are studied. An evaluation of new experimental data from single DNA molecules labeled with a fluorescent conformation probe proves the heterogeneity of the ensemble system. Also, the anomalicity is concluded to be low for single events. The evaluation method can be applied in other experiments where two-state reversible processes are studied for isolated systems.

Interferometry and Fluorescence Detection for Simultaneous Analysis of Labeled and Unlabeled Nanoparticles in Solution

A novel fluctuation spectroscopy technique based on interferometry is described. The technique, termed scattering interference correlation spectroscopy (SICS), autocorrelates the signals from the forward-scattered and transmitted laser light from nanoparticles (NPs) in solution. SICS has two important features: First, for unlabeled NPs with known refractive index, it analyzes not only the diffusion coefficient but also the effective cross section and concentration in a single measurement. Second, it can be combined with fluorescence correlation spectroscopy (FCS) for simultaneous analysis of labeled and unlabeled NPs. SICS is here demonstrated on unlabeled M13 phages and on unlabeled NPs with diameters of 210 nm down to 26 nm. It is also shown how the combination of SICS and FCS can be used to determine the fraction of fluorescent NPs in a mixture and estimate K(d) from a single binding measurement.

Highly Sensitive FRET-FCS Detects Amyloid β-Peptide Oligomers in Solution at Physiological Concentrations

Oligomers formed by the amyloid β-peptide (Aβ) are pathogens in Alzheimers disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, Aβ oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of Förster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of Aβ, FRET-FCS detected oligomers consisting of less than 10 Aβ molecules, which coexisted with the monomers at fractions as low as 2 ± 2%. Thus, we demonstrate for the first time direct detection of small fractions of Aβ oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

Analysis of the RNase T1 mediated cleavage of an immobilized gapped heteroduplex via fluorescence correlation spectroscopy.

Abstract We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine Blabeled substrate by ribonuclease T1. A gapped heteroduplex substrate a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides was immobilized via biotin to a streptavidincoated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10[-21] mol 0.05 f of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).