Single-molecule high-resolution imaging with photobleaching

Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a “gap” in the ability of optical techniques to measure distances on the 10- to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10–20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms.     

Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

Polar molecules align in electric fields when the dipole energy (proportional to field intensity E × dipole moment p) exceeds the thermal rotational energy. Small molecules have low p and align only at inordinately high E or upon extreme cooling. Many biomacromolecules and ions are strong permanent dipoles that align at E achievable in gases and room temperature. The collision cross-sections of aligned ions with gas molecules generally differ from orientationally averaged quantities, affecting ion mobilities measured in ion mobility spectrometry (IMS). Field asymmetric waveform IMS (FAIMS) separates ions by the difference between mobilities at high and low E and hence can resolve and identify macroion conformers based on the mobility difference between pendular and free rotor states. The exceptional sensitivity of that difference to ion geometry and charge distribution holds the potential for a powerful method for separation and characterization of macromolecular species. Theory predicts that the pendular alignment of ions in gases at any E requires a minimum p that depends on the ion mobility, gas pressure, and temperature. At ambient conditions used in current FAIMS systems, p for realistic ions must exceed ≈300–400 Debye. The dipole moments of proteins statistically increase with increasing mass, and such values are typical above ≈30 kDa. As expected for the dipole-aligned regime, FAIMS analyses of protein ions and complexes of ≈30–130 kDa show an order-of-magnitude expansion of separation space compared with smaller proteins and other ions.     

Metastability transfer spectroscopy with two like ions in the same trap

High-resolution optical spectroscopy of an individual trapped ion is hampered by lack of sharp lasers. This suggests the use of a second metastable excited ion as an ultrasharp light source. To this end, laser-cool two barium ions to an equilibrium distance of ≈8 μm on the z (symmetry) axis of the trap and, in this (earth)(Ba⁺)₂-molecule, visually or photoelectrically identify them as A and B by their location. Briefly turn on a 455-nm spectral lamp until one of the ions, say the A ion, is pumped into the metastable D_5/2 level and turns invisible. Focus on the visible, spatially well-resolved B ion and turn off the blue and red illumination lasers for ≈15 s. Then turn them back on again and check on whether the excitation by chance has been transferred to the B ion and is now in the D_5/2 level and dark while the A ion is bright. The cross section for absorption of the λ(D_5/2 → S_1/2) λ₀ = 1.76 μm radiation by a stationary ion can be >λ₀²/2π. Thus, by pushing the two ions together to ≈λ₀/4 by turning on a much stronger trapping field during the excitation exchange period, one might be able to detect excitation transfer in >10% of the attempts. The ions are tuned relative to each other by a 0- to 10-mV/cm variable dc field in the z direction, which displaces them axially and causes them to see different rf fields, which Stark-shifts their frequencies. In this way, a resonant transfer response as sharp as twice the natural width of the D_5/2 level, 11 mHz or a Q ≈ 0.4 × 10¹⁷, might be demonstrated.     

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than ≈100 μs and distances greater than ≈2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-μs decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type (“D_phys”). D_phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the “speed limit” of folding. D_phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.     

The dynamics of structural deformations of immobilized single light-harvesting complexes

Single assemblies of the intact light-harvesting complex LH2 from Rhodopseudomonas acidophila were bound to mica surfaces at 300 K and examined by observing their fluorescence after polarized light excitation. The complexes are generally not cylindrically symmetric. They act like elliptic absorbers, indicating that the high symmetry found in crystals of LH2 is not present when the molecules are immobilized on mica. The ellipticity and the principal axes of the ellipses fluctuate on the time scale of seconds, indicating that there is a mobile structural deformation. The B850 ring of cofactors shows significantly less asymmetry than B800. The photobleaching strongly depends on the presence of oxygen.     

Fluorescence lifetimes in the bipartite model of the photosynthetic apparatus with alpha, beta heterogeneity in photosystem II

Recent studies of the lifetime of fluorescence after picosecond pulse excitation of photosynthetic organisms revealed relatively complex decay kinetics that indicated a sum of three exponential components with lifetimes spanning the range from about 0.1-2.5 ns. These fluorescence lifetime data were examined in the context of a simple photochemical model for photosystem II that was used previously to account for fluorescence yield data obtained during continuous illumination. The model, which consists of a single fluorescing species of antenna chlorophyll and a reaction center, shows that, in general, the decay kinetics after pulse excitation should consist of the sum of two exponential decays. The model also shows that in going from open to closed reaction centers the lifetime of fluorescence may increase much more than the yield of fluorescence and surprisingly long fluorescence lifetimes can be obtained. However, conditions can be stated where fluorescence will decay essentially as a single component and with lifetime changes that are proportional to the yield changes. A heterogeneity was also introduced to distinguish photosystem II_α units, which can transfer excitation energy among themselves but not the photosystem I, and photosystem II_β units, which can transfer energy to photosystem I but not to other photosystem II units. It is proposed that the rather complex fluorescence lifetime data can be accounted for in large part by the simple photochemical model with the α, β heterogeneity in photosystem II.     

Fluorescence energy transfer studies on the active site of papain

Measurements have been performed of the excited-state lifetimes and fluorescence yields of papain tryptophan units when acyl derivatives of Phe-glycinal are bound at the active site of the enzyme. The enhancement of tryptophan fluorescence in complexes of papain with the acetyl or benzyloxycarbonyl derivatives is not stereospecific with respect to the configuration of the phenylalanyl residue, and the L and D isomers are equally effective as active-site-directed inhibitors of papain action. Evidence is offered in favor of the conclusion that this enhancement is primarily a consequence of the interaction of the phenylalanyl side chain of the inhibitor with Trp-69 of the enzyme. This residue can exchange fluorescence energy with the other four tryptophans of papain (Trp-7, Trp-26, Trp-177, Trp-181) upon excitation near their absorption maxima, but such “homotransfer” is absent if they are excited at the long-wave edge of their absorption spectra. Crystallographic data indicate that Trp-26 is most favorably positioned for efficient energy exchange with Trp-69, and the fluorescence data have been used to calculate a distance of 11 Å between the two residues; this value is in satisfactory agreement with that found by crystallography. When derivatives of Phe-glycinal bearing an amino-terminal mansyl [6-(N-methylanilino)-2-naphthalene sulfonyl] group are bound at the active site of papain, the tryptophan fluorescence is quenched, as compared with that of the complex of papain with acetyl-Phe-glycinal, indicating energy transfer from papain tryptophan (most probably via Trp-26) to the fluorescent probe group. Although the L and D isomers of mansyl-Phe-glycinal are equally effective as inhibitors of papain action, the fluorescence quenching by the two isomers is different.     

Unliganded gating of acetylcholine receptor channels

We estimated the unliganded opening and closing rate constants of neuromuscular acetylcholine receptor-channels (AChRs) having mutations that increased the gating equilibrium constant. For some mutant combinations, spontaneous openings occurred in clusters. For 25 different constructs, the unliganded gating equilibrium constant (E₀) was correlated with the product of the predicted fold-increase in the diliganded gating equilibrium constant caused by each mutation alone. We estimate that (i) E₀ for mouse, wild-type α₂βδε AChRs is ≈1.15 × 10⁻⁷; (ii) unliganded AChRs open for ≈80 μs, once every ≈15 min; (iii) the affinity for ACh of the O(pen) conformation is ≈10 nM, or ≈15,600 times greater than for the C(losed) conformation; (iv) the ACh-monoliganded gating equilibrium constant is ≈1.7 × 10⁻³; (v) the C→O isomerization reduces substantially ACh dissociation, but only slightly increases association; and (vi) ACh provides only ≈0.9 k_BT more binding energy per site than carbamylcholine but ≈3.1 k_BT more than choline, mainly because of a low O conformation affinity. Most mutations of binding site residue αW149 increase E₀. We estimate that the mutation αW149F reduces the ACh affinity of C only by 13-fold, but of O by 190-fold. Rate–equilibrium free-energy relationships for different regions of the protein show similar slopes (Φ values) for un- vs. diliganded gating, which suggests that the conformational pathway of the gating structural change is fundamentally the same with and without agonists. Agonist binding is a perturbation that (like most mutations) changes the energy, but not the mechanism, of the gating conformational change.     

Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll–protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to ≈60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron–nuclear–nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from ≈2:1 to ≈5:1 in the mutants compared with ≈2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation–reduction midpoint potential.     

Conformational isomerization kinetics of pent-1-en-4-yne with 3,330 cm-1 of internal energy measured by dynamic rotational spectroscopy

We demonstrate the application of molecular rotational spectroscopy to measure the conformation isomerization rate of vibrationally excited pent-1-en-4-yne (pentenyne). The rotational spectra of single quantum states of pentenyne are acquired by using a combination of IR–Fourier transform microwave double-resonance spectroscopy and high-resolution, single-photon IR spectroscopy. The quantum states probed in these experiments have energy eigenvalues of ≈3,330 cm⁻¹ and lie above the barrier to conformational isomerization. At this energy, the presence of intramolecular vibrational energy redistribution (IVR) is indicated through the extensive local perturbations found in the high-resolution rotation–vibration spectrum of the acetylenic C–H stretch normal-mode fundamental. The fact that the IVR process produces isomerization is deduced through a qualitatively different appearance of the excited-state rotational spectra compared with the pure rotational spectra of pentenyne. The rotational spectra of the vibrationally excited molecular eigenstates display coalescence between the characteristic rotational frequencies of the stable cis and skew conformations of the molecule. This coalescence is observed for quantum states prepared from laser excitation originating in the ground vibrational state of either of the two stable conformers. Experimental isomerization rates are extracted by using a three-state Bloch model of the dynamic rotational spectra that includes the effects of chemical exchange between the stable conformations. The time scale for the conformational isomerization rate of pentenyne at total energy of 3,330 cm⁻¹ is ≈25 ps and is 50 times slower than the microcanonical isomerization rate predicted by the statistical Rice–Ramsperger–Kassel–Marcus theory.     

The roles of specific xanthophylls in photoprotection

Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the α- or β-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (α-carotene branch), zeaxanthin, and antheraxanthin (β-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), α-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.     

Relativistic cyclotron resonance shape in magnetic bottle geonium

The thermally excited axial oscillation of the electron through the weak magnetic bottle needed for the continuous Stern-Gerlach effect modulates the cyclotron frequency and produces a characteristic ≈ 12-kHz-wide vertical rise-exponential decline line shape of the cyclotron resonance. At the same time the relativistic mass shift decreases the frequency by ≈ 200 Hz per cyclotron motion quantum level n. Nevertheless, our analysis of the complex line shape shows that it should be possible to produce an abrupt rise in the cyclotron quantum number n from 0 to ≈ 20 over a small fraction of 200 Hz, when the 160-GHz microwave drive approaches the n = 0 → 1 transition, and a jump of 14 levels over a frequency increment of 200 Hz has already been observed in preliminary work. This realizes an earlier proposal to generate a very sharp cyclotron resonance feature by quasithermal excitation with a square noise band and should provide a way to detect spin flips when a weak bottle is used to reduce the broadening of the g - 2 resonance by a factor of 20.     

Survey of large protein complexes in D. vulgaris reveals great structural diversity

An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr ≈ 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate ≈10 times greater than that of previous “proteomic” screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.     

Principles of quasi-equivalence and Euclidean geometry govern the assembly of cubic and dodecahedral cores of pyruvate dehydrogenase complexes

The pyruvate dehydrogenase multienzyme complex (M_r of 5–10 million) is assembled around a structural core formed of multiple copies of dihydrolipoyl acetyltransferase (E2p), which exhibits the shape of either a cube or a dodecahedron, depending on the source. The crystal structures of the 60-meric dihydrolipoyl acyltransferase cores of Bacillus stearothermophilus and Enterococcus faecalis pyruvate dehydrogenase complexes were determined and revealed a remarkably hollow dodecahedron with an outer diameter of ≈237 Å, 12 large openings of ≈52 Å diameter across the fivefold axes, and an inner cavity with a diameter of ≈118 Å. Comparison of cubic and dodecahedral E2p assemblies shows that combining the principles of quasi-equivalence formulated by Caspar and Klug [Caspar, D. L. & Klug, A. (1962) Cold Spring Harbor Symp. Quant. Biol. 27, 1–4] with strict Euclidean geometric considerations results in predictions of the major features of the E2p dodecahedron matching the observed features almost exactly.     

Structural properties of double-stranded RNAs associated with biological control of chestnut blight fungus

Double-stranded RNAs (ds RNAs) are thought to be the cytoplasmic determinants responsible for the phenomenon of transmissible hypovirulence in the chestnut blight fungus Endothia parasitica [Murr.] Anderson. The three major ds RNA components associated with the North American hypovirulent strain, Grand Haven 2, were characterized with respect to molecular-hybridization specificity and RNase T1-digestion patterns. The large (L-RNA; ≈9 kilobase pairs) and middle-sized (M-RNA; ≈3.5 kilobase pairs) ds RNA components cross-hybridized under stringent conditions and exhibited indistinguishable partial and complete RNase T1 digestion patterns relative to their 5′ and 3′ termini. These results suggest that M-RNA was derived from L-RNA by an internal deletion event. The small (S-RNA; ≈1 kilobase pair) RNA was unrelated to L- and M-RNA by these criteria. However, all three ds RNA components contained RNase T1-resistant oligonucleotides at one 5′ terminus and at the corresponding 3′ terminus of the complementary strand. These RNase T1-resistant species exhibited properties consistent with stretches of poly(uridylic acid) and poly(adenylic acid), respectively. The combined results are discussed in terms of the structural organization of hypovirulence-associated ds RNA molecules and their similarities to “double-stranded” RNA molecules observed in plant and animal cells infected with single-stranded RNA viruses.     

Giant circular dichroism of high molecular weight chlorophyllide-apomyoglobin complexes

Chlorophyllide a and the apoprotein of myoglobin (Mb) spontaneously form three types of complex. The M (M_r ≈ 3 × 10⁵) and H (M_r ≥ 4 × 10⁶) complexes, but not the L (M_r ≈ 1.7 × 10⁴), display a circular dichroism (CD) spectrum that is highly red-shifted, nonconservative, and very intense—characteristics shared by the CD spectra of reaction center complexes from purple photosynthetic bacteria. At its 710-nm peak, the H complex CD spectrum has a larger magnitude, 0.06 differential absorbance per unit total absorbance, than has been reported for chlorophyll in any medium.     

Morphological, physiological, and biochemical changes in rhodopsin knockout mice

Mutations in rod opsin, the visual pigment protein of rod photoreceptors, account for ≈15% of all inherited human retinal degenerations. However, the physiological and molecular events underlying the disease process are not well understood. One approach to this question has been to study transgenic mice expressing opsin genes containing defined mutations. A caveat of this approach is that even the overexpression of normal opsin leads to photoreceptor cell degeneration. To overcome the problem, we have reduced or eliminated endogenous rod opsin content by targeted gene disruption. Retinas in mice lacking both opsin alleles initially developed normally, except that rod outer segments failed to form. Within months of birth, photoreceptor cells degenerated completely. Retinas from mice with a single copy of the opsin gene developed normally, and rods elaborated outer segments of normal size but with half the normal complement of rhodopsin. Photoreceptor cells in these retinas also degenerated but did so over a much slower time course. Physiological and biochemical experiments showed that rods from mice with a single opsin gene were ≈50% less sensitive to light, had accelerated flash-response kinetics, and contained ≈50% more phosducin than wild-type controls.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from ≈50 to ≈300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented ≈6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

Primary electron transfer reactions in modified reaction centers from Rhodopseudomonas sphaeroides

Absorption spectra were measured by means of an optical multichannel analyzer in Rhodopseudomonas sphaeroides R-26 reaction centers (RCs) modified by treatment with NaBH₄ at various times (≥1 ps) after the onset of a short excitation flash at 880 nm. Most of these RCs (75-95%) have only one “monomeric” bacteriochlorophyll-800 (B₁) molecule and are as active as the original RCs. The duration of the excitation and measuring pulses was ≈33 ps. If the center of the excitation pulse preceded the center of the measuring pulse by 36-40 ps, the formation of a state P^E (early state), which is converted to the state P^F (P⁺ bacteriopheophytin^-) in 4 ± 1 ps (1/e time), was observed. Also the kinetics and the spectrum of the stimulated emission (reflecting the kinetics and the emission spectrum of the excited state P^*) were determined. The difference spectrum of the state P^E approximately equals the sum of the spectra of the states P^* (≈65%) and ¹[P⁺B₁^-] (≈35%). This indicates that B₁^- is an intermediate in the electron transfer from P^* to bacteriopheophytin, H₁, transferring this electron with a rate constant of (4 × 0.35 ps)^-1 = 7 × 10¹¹ s^-1.