Fluorescence Polarization Data Research Articles

Myosin lever arm orientation in muscle determined with high angular resolution using bifunctional spin labels.

Despite advances in x-ray crystallography, cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain (LCD; lever arm) under ambient conditions in vertebrate muscle. Here, we measure the orientation of LCD elements in demembranated muscle fibers by electron paramagnetic resonance (EPR) using a bifunctional spin label (BSL) with an angular resolution of 4°. To achieve stereoselective site-directed labeling with BSL, we engineered a pair of cysteines in the myosin regulatory light chain (RLC), either on helix E or helix B, which are roughly parallel or perpendicular to the myosin lever arm, respectively. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtain EPR spectra from which the angular distributions of BSL, and thus the lever arm, can be determined with high resolution relative to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibits both ordered (σ ∼9-11°) and disordered (σ > 38°) populations. Using these angles to determine the orientation of the lever arm (LCD combined with converter subdomain), we observe that the oriented population corresponds to a lever arm that is perpendicular to the muscle fiber axis and that the addition of ATP in the absence of Ca2+ (inducing relaxation) shifts the orientation to a much more disordered orientational distribution. Although the detected orientation of the myosin light chain lever arm is ∼33° different than predicted from a standard "lever arm down" model based on cryo-EM of actin decorated with isolated myosin heads, it is compatible with, and thus augments and clarifies, fluorescence polarization, x-ray interference, and EM data obtained from muscle fibers. These results establish feasibility for high-resolution detection of myosin LCD rotation during muscle contraction.

Implementation of an experimental and computational tool set to study protein-mAb interactions.

This work focused on the development of a combined experimental and computational tool set to study protein-mAb interactions. A model protein library was first screened using cross interaction chromatography to identify proteins with the strongest retention. Fluorescence polarization was then employed to study the interactions and thermodynamics of the selected proteins-lactoferrin, pyruvate kinase, and ribonuclease B with the mAb. Binding affinities of lactoferrin and pyruvate kinase to the mAb were seen to be relatively salt insensitive in the range examined. Further, a strong entropic contribution was observed, suggesting the importance of hydrophobic interactions. On the other hand, ribonuclease B-mAb binding was seen to be enthalpically driven and salt sensitive, indicating the importance of electrostatic interactions. Protein-protein docking was then carried out and the results identified the CDR region on the mAb as an important binding site for all three proteins. The binding interfaces identified for the mAb-lactoferrin and mAb-pyruvate kinase systems were found to contain complementary hydrophobic and oppositely charged clusters on the interacting regions which were indicative of both hydrophobic and electrostatic interactions. On the other hand, the binding site on ribonuclease B was predominantly positively charged with minimal hydrophobicity. This resulted in an alignment with negatively charged clusters on the mAb, supporting the contention that these interactions were primarily electrostatic in nature. Importantly, these computational results were found to be consistent with the fluorescence polarization data and this combined approach may have utility in examining mAb-HCP interactions which can often complicate the downstream processing of biologics. © 2019 American Institute of Chemical Engineers.

Measuring fluorescence polarization with a dichrometer

A method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of a polarizer.

Structure-Activity Relationships of Cbx7 Inhibitors, Including Selectivity Studies against Other Cbx Proteins.

The five human polycomb (Pc) paralog proteins, chromobox homolog (Cbx) 2/4/6/7/8, are a family of chromodomain containing methyllysine reader proteins that are canonical readers of trimethyllysine 27 on histone 3 (H3K27me3). The aberrant expression of the Cbx7 gene is implicated in several cancers including prostate, gastric, thyroid, pancreas, and colon cancer. Previous reports on antagonizing the molecular recognition of Cbx7–H3K27me3 with chemical inhibitors showed an impact on prostate cancer cell lines. We report here on the design, synthesis, and structure–activity relationships of a series of potent peptidomimetic antagonists that were optimized on a trimethyllysine-containing scaffold to target Cbx7. The ligands were characterized using fluorescence polarization (FP) for their binding efficiency and selectivity against the Pc paralog Cbx proteins. The most selective ligand 9, as indicated by the FP data analysis, was further characterized using the isothermal titration calorimetry (ITC). Compound 9 exhibits a 220 nM potency for Cbx7 and exhibits 3.3, 1.8, 7.3 times selective for Cbx7 over Cbx2/4/8 and 28-fold selective over the HP1 family member Cbx1. Our research provides several potent and partially selective inhibitors for Cbx2/4/7 that do not contain trimethyllysine. Our models and binding data suggest that the aromatic cages of Cbx7/Cbx4 can accommodate larger alkyl groups such as diisobutyl substitution on the lysine nitrogen.

Chlorophyll a and chlorophyllide a inside liposomes made of saturated and unsaturated lipids: A possible impact of the lipids microenvironment

The aim of this work was to examine a possible impact of liposomes lipids microenvironment, dictated by a chemical composition of the fatty acid branches, on incorporation and spectral behaviour of chlorophyll a, and its derivative, chlorophyllide a inside small liposomes. The liposomes with the incorporated chlorophylls were made of dimirystoyl phosphatidylcholine (DMPC), and unsaturated phosphatidylcholine (PC), containing significant fractions of unsaturated fatty acid moieties. In order to achieve the goal, both absorption and fluorescence polarization spectroscopy were applied, and the obtained data for the two incorporated pigments, which play a role of molecular sensors, were compared. In addition, quercetin, a well-known antioxidant, was used as the (chlorophylls) emission quencher, in order to estimate the type of environment sensed by the two pigments for the two liposomes that differ in chemical composition. The results, based primarily on fluorescence polarization data have shown that the emissions as well as the emission quenching were notably affected by a change in the lipids? chemical composition. That is an indirect proof of the impact of the liposomes microenvironment on the incorporated pigments? spectral behaviour.

Denaturation of human serum albumin under the action of cetyltrimethylammonium bromide according to fluorescence polarization data of protein

Denaturation of human serum albumin (HSA) under the action of cationic detergent cetyltrimethylammonium bromide (CTAB) is studied at different pH values by estimating the rotational diffusion of protein via fluorescence polarization. The degree of polarization of HSA tryptophan fluorescence, the rotational relaxation time, the rotational diffusion coefficient and the effective Einstein radius of the HSA molecules in solutions with different CTAB concentrations at different pH values are determined. The obtained rotational diffusion parameters of the HSA molecules show that under the action of CTAB, HSA denaturation has a one-stage character and proceeds more intensely and effectively at pH values higher than the pI value of protein (4.7).

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: A biophysical approach to transfection efficiency

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C14H29(CH3)2N+(CH2)2N+(CH3)2C14H29]2Br− and herein designated 14–2–14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14–2–14, 14–2–14:Chol (1:1 molar ratio) and 14–2–14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/− charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14–2–14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/− charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.

Spectroscopic Detection of Tetracationic Porphyrin H-Aggregation on Polyanionic Matrix of Inorganic Polyphosphate

Self-assembly of tetracationic porphyrin TMPyP(4+) onto polyanionic matrix of inorganic polyphosphate (PPS) in aqueous solutions has been studied in a wide range of molar phosphate-to-dye ratios using techniques of polarized fluorescence, absorption, resonance Raman spectroscopy and static light scattering. The binding of TMPyP(4+) to PPS is characterized by the binding constant of 3 x 10(5) M(-1) and the cooperativity parameter of about 150. The fluorescence quenching of the bound TMPyP(4+) evidences the stacking of the porphyrine chromophores. Under the stoichiometric binding ratio TMPyP(4+) forms extended continuous face-to-face aggregates (so-called H-aggregates) which manifest themselves by a blue shift (12 nm) and a large hypochromisity (51%) of the Soret absorption band. Each face-to-face TMPyP(4+) stack is formed with participation of four PPS chains. Formation of such columnar aggregates is promoted by the ability of PPS chains to take a helix conformation where negative charges are arranged along two oppositely situated rows with intercharge distance of 0.36 nm which corresponds to the thickness of the porphyrin pi-electronic system. The ability of each PPS strand to be template for formation of two porphyrin stacks results in the integration of the adjacent stacks into higher-order aggregates which dimension was estimated from the fluorescence polarization data.

Fluorescent Indicators Distributed throughout the Pharmacophore of Cholecystokinin Provide Insights into Distinct Modes of Binding and Activation of Type A and B Cholecystokinin Receptors

Ligand probes with fluorescent indicators positioned throughout the pharmacophoric domain can provide important insights into the molecular basis of receptor binding and activation as reflected in the microenvironment of each indicator while docked at a receptor. We developed three cholecystokinin-like probes with Aladan situated at the N terminus, in the mid-region, and at the C terminus (positions 24, 29, and 33, respectively). These were studied in solution and docked at type A and B cholecystokinin receptors. This study demonstrated clear differences in mechanisms of cholecystokinin binding and activation of these structurally related receptors with distinct agonist structure-activity relationships. The fluorescence characteristics of Aladan are highly sensitive to the polarity of its microenvironment. The mid-region probe was least accessible to the aqueous milieu as determined by fluorescence emission spectra and iodide quenching, which was not altered by changes in conformation from active to inactive. Accessibility of the N- and C-terminal probes was affected by receptor conformation. The position 24 probe was more easily quenched in the active than in the G protein-uncoupled conformation for both receptors. However, the position 33 probe docked at the type A cholecystokinin receptor was more easily quenched in the active conformation, whereas the same probe docked at the type B cholecystokinin receptor was more easily quenched in the inactive conformation. Fluorescence anisotropy and red edge excitation shift determinations confirmed these observations and supported the proposed movements. Although both type A and B cholecystokinin receptors bind cholecystokinin with high affinity, resulting in fully efficacious biological responses, these receptors utilize distinct molecular modes of binding.

Detection of single nucleotide polymorphisms through the application of lifetime fluorescent probes

Fluorescence-based approaches for genotyping single nucleotide polymorphisms (SNPs) commonly involve enzymatic cleavage, stringent control of hybridization temperature, or extensive post amplification clean up. Our research to SNP genotyping employs fluorescence lifetime probes, which consist of a single fluorophore that is covalently bound to a oligonucleotide complementary to a SNP allele. We require that the matched and mismatched forms correspond to different lifetimes of the fluorophore. In other words, fluorophore lifetime reports whether the base has entered into the double strand or is mismatched. The constraints on hybridization conditions are greatly relaxed and enzymatic cleavage is not required. This fluorescent lifetime-based technology allows resolving SNP alleles in both the homozygous and heterozygous states using a single lifetime probe. Initial studies have been conducted on the human β-globin gene, known to cause sickle cell anemia. Our results show the fluorophore lifetime is measurably different for the mutant and wild forms. We are currently characterizing the effects of probe/target base pair mismatch beyond the initial SNP. Preliminary studies suggest fluorescence polarization data likely provides additional specificity. This research was supported by the North Dakota INBRE grant.

A fluorescence polarization study of the O2+ A2Πu → X2Πg and the b4Σg− → a4Πu transitions in the excitation range 17–25 eV

The fluorescence yields and polarizations of the O2+ A2Πu → X2Πg and the b4Σg− → a4Πu transitions have been measured between their thresholds and 25 eV using synchrotron radiation. The O2+ ions may be formed either through direct photoionization or through excitation into a Rydberg state followed by autoionization. The alignment of the resulting (A2Πu or b4Σg− state) ion has been determined by measuring the degree of linear polarization of the fluorescence emitted in the subsequent decay. The fluorescence yields and polarizations display prominent structure associated with autoionizing Rydberg states belonging to series converging onto the b4Σg−, B2Σg− or the c4Σu− ionization thresholds. The structure in the fluorescence polarization data has enabled the symmetry of the super-excited states to be deduced. Although the interpretation of this symmetry information has been straight-forward in regard to the Rydberg series converging onto the c4Σu− limit, the situation is less clear for the Rydberg series converging onto the b4Σg− or the B2Σg− limits. The measured polarizations have been compared with calculated values and, even for the A2Πu → X2Πg transition where the theoretical model incorporates resonant processes, the agreement is not particularly good.

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: Implication in hemodialysis mediated atherogenic LDL modification

Objective: Al3+ stimulates Fe2+ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al3+ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al3+ on LDL oxidation.Materials and methods: Using different LDL modifying systems (Fe2+, Cu2+, free radical generating compounds, human endothelial cells, hemin/H2O2 and HOCl), the influence of Al3+ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.Results: Al3+ could stimulate the oxidizability of LDL by Fe2+, but not in the other systems tested. Al3+ and Fe2+ were found to bind to LDL and Al3+could compete with Fe2+ binding to the lipoprotein. Fluorescence polarization data indicated that Al3+ does not affect the phospholipid compartment of LDL.Conclusions:The results indicate that increased LDL oxidation by Fe2+ in presence of Al3+ might be due to blockage of Fe2+ binding sites on LDL making more free Fe2+ available for lipid oxidation.

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin1Areceptor

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT(1A) receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT(1A) receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT(1A) receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT(1A) receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT(1A) receptor function.

Fluorescence anisotropy as a probe to study tracer proteins in crowded solutions.

Fluorescence anisotropy has been widely used to study the dynamics and interactions of biomolecules in diluted solutions. Comparable studies on single tracer macromolecules at the cellular level are now feasible because of the recent development of non-invasive fluorescence markers, like the growing family of the green fluorescence proteins (GFPs), and the advances in time-resolved fluorescence microscopy instrumentation. The interpretation of fluorescence polarization data in terms of dynamics and biological function of the macromolecular complexes in these physiological environments requires a deep understanding of the tracer rotational diffusion in such complex media. In this work we have studied the rotational diffusion of a tracer protein, apomyoglobin labeled with 1-anilino-8-naphthalene sulfonate, in crowded solutions of an unrelated protein, ribonuclease A. We have evaluated the deviation of the different tracer rotational motions from the Stokes-Einstein-Debye diffusion behavior, and its relation to the properties of the transient molecular cavities where the tracer is rotating in the fluorescence lifetime window. Finally, we have analyzed the application of fluorescence polarization methods to determine the apparent equilibrium constants of homo and hetero-associations of macromolecules in crowded conditions.

Toxin Binding of Tolevamer, a Polyanionic Drug that Protects against Antibiotic-Associated Diarrhea

Tolevamer, (GT160-246), is a sodium salt of styrene sulfonate polymer that is under development for the treatment of diarrhea caused by infection with Clostridium difficile. Pulsed ultrafiltration binding experiments in phosphate buffer containing 0.15 M Na + provide per polymer chain dissociation constants of 133 nM and 8.7 μM for the binding of tolevamer to C. difficile toxins A and B, respectively. At 0.05 M Na +, the binding of toxin A to tolevamer is irreversible, whereas the dissociation constant to toxin B under these conditions is 120 nM. Binding constants obtained from fluorescence polarization data for toxin A binding to tolevamer at 0.15 M Na + agree substantially with those obtained by pulsed ultrafiltration. The binding activity of tolevamer reported here correlates well with previously reported results for the inhibition of the biological activity of C. difficile toxins A and B. From the fluorescence polarization data, it is estimated that one toxin A molecule interacts with between 600 to 1000 monomer units on tolevamer at 0.15 M Na +. Thus, the data suggest a very large interaction surface between polymer and toxin A.

Effects of unsaturated fatty acids and triacylglycerols on phosphatidylethanolamine membrane structure

Lipid intake in diet regulates the membrane lipid composition, which in turn controls activities of membrane proteins. There is evidence that fatty acids (FAs) and triacylglycerols (TGs) can alter the phospholipid (PL) mesomorphism. However, the molecular mechanisms involved are not fully understood. This study focuses on the effect of the unsaturation degree of the C-18 FAs, oleic acid (OA), linoleic acid and linolenic acid, and their TGs, triolein (TO), trilinolein, and trilinolenin, on the structural properties of phosphoethanolamine PLs. By means of X-ray diffraction and 31P-NMR spectroscopy, it is shown that both types of molecules stabilize the HII phase in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) liposomes. Several structural factors are considered to explain the correlation between the FA unsaturation degree and the onset temperature of the HII phase. It is proposed that TGs could act as lateral spacers between polar DEPE groups, providing an increase in the effective surface area per lipid molecule that would account for the structural parameters of the HII phase. Fluorescence polarization data indicated a fluidification effect of OA on the lamellar phase. TO increased the viscosity of the hydrophobic core with a high effect on the HII phase.

Decorin Binds Fibrinogen in a Zn2+-dependent Interaction

We have previously shown that decorin, a member of the small leucine-rich proteoglycan family of extracellular matrix proteoglycans/glycoproteins is a Zn(2+) metalloprotein at physiological Zn(2+) concentrations (Yang, V. W-C., LaBrenz, S. R., Rosenberg, L. C., McQuillan, D., and Höök, M. (1999) J. Biol. Chem. 274, 12454-12460). We now report that the decorin proteoglycan binds fibrinogen in the presence of Zn(2+). The fibrinogen-binding site is located in the N-terminal domain of the decorin core protein and a 45-amino acid peptide representing this domain binds to the fibrinogen D fragment with an apparent K(D) of 1.7 x 10(-6) m, as determined from fluorescence polarization data. Furthermore, we show that Zn(2+) promotes the self-association of decorin. The N-terminal domain of the core protein also mediates this activity. The results of solid-phase binding assays and gel filtration chromatography suggest that the N-terminal domain of decorin, when present at low micromolar concentrations, forms an oligomer in a Zn(2+)-dependent manner. Thus, Zn(2+) appears to play a pivotal role in the interactions and biological function of decorin.

Interaction of Ochratoxin A with Human Serum Albumin. A Common Binding Site of Ochratoxin A and Warfarin in Subdomain IIA

Optical spectroscopy was used to examine the binding of ochratoxin A (OTA) and warfarin (WAR) to human serum albumin (HSA). Both molecules in the deprotonated form showed high affinity binding to HSA. The close proximity of the highest affinity binding site of the OTA dianion to that of the WAR monoanion was suggested by depolarization of WAR emission in ternary mixtures with [OTA]/[WAR] ≥ 3. Fluorescence polarization data also showed that both OTA and WAR simultaneously bind to HSA for 0.1 ≤ [OTA]/[WAR] ≤ 1. The failure of WAR to displace OTA under these conditions is in accord with the much smaller binding constant for WAR. In all displacement experiments either the HSA-to-WAR or HSA-to-OTA concentration ratio was kept constant and close to unity. Evidence of energy transfer from electronically excited WAR to OTA when both species are bound to HSA was obtained from fluorescence emission data. The efficiency of this energy transfer provided an estimate of 27 Å as the upper limit of the distance between WAR and OTA. WAR bound to HSA strongly quenched the fluorescence of the single tryptophan residue, Trp214. However, the quenching mechanism was different from the energy transfer mechanism observed for quenching of WAR by OTA. The results show that OTA and WAR share a common binding site in subdomain IIA, with OTA having a higher binding affinity. In addition, WAR can occupy another binding site on HSA when OTA is bound. The data suggest that a secondary binding site of OTA is located in domain III, while that of WAR is in domain I.

Adenine-Thymine Base Pair Recognition by an Anthryl Probe from the DNA Minor Groove

Synthesis and DNA binding properties of geometric isomers, 9(3-aminopropyl)anthracene (APAC) and 9(N-ethyl)aminomethyl anthracene (N-Et-AMAC) are reported here. The aminopropyl side chain of APAC can position the amino function in the DNA grooves for hydrogen bonding with the heterocyclic bases. In contrast, the N-ethyl-aminomethyl side chain of N-Et-AMAC is too short to make contacts in the grooves. The DNA binding properties are examined using absorption, fluorescence, circular dichroism (CD), singlet–singlet energy transfer, and DNA melting experiments. In addition to calf thymus DNA (CTDNA), we examined poly(dA-dT), poly(dG-dC), poly(dA)-poly(dT), and poly(dI-dC). The spectroscopic and thermal studies indicate intercalation of the anthryl probes into the helix. The binding properties, however, depended on the side chain as well as the DNA sequence. The binding of APAC to poly(dA-dT) is preferred 3.4 times better over poly(dG-dC) while binding of N-Et-AMAC, the geometric isomer of APAC, is nearly the same with all the DNA polymers examined. Hydrogen bonding of the probe side chain from the minor groove of the helix is tested by using inosine-cytosine sequences. Consistent with the above data, APAC binds to poly(dI-dC) with binding constants similar to that of poly(dA-dT) sequences. Thus, both chemical and biochemical methods are used to test the hydrogen bonding of the probe side chains with the helix. The DNA sequence discrimination observed here clearly indicates the position of the amino function on the side chain plays an important role in DNA sequence recognition. The helix melting temperature of CTDNA is increased from 70°C to 80 and 82°C by APAC and N-Et-AMAC, respectively, indicating the helix stabilization upon intercalation of the probes. Intercalation is also supported by the CD spectra of the probe-DNA complexes. The probes are achiral and binding to the helix leads to induced CD spectra in the 300–400nm region, away from the DNA absorption bands. The positive CD bands observed indicate intercalation of the anthryl chromophore with its long axis perpendicular to the base-pair dyad axis. Fluorescence polarization data clearly support intercalation of both probes into the above sequences, including poly(dI-dC), except that no polarization was observed with APAC bound to poly(dG-dC). This result is consistent with preferential binding of APAC to AT sites over GC sites and efficient quenching of anthryl emission at GC sites. Excitation into the DNA absorption bands results in sensitized emission from the anthryl probes and the efficiency of energy transfer depends upon the sequence. While AT sites sensitize anthryl emission efficiently, no sensitization was noted for GC sequences. These data clearly show that GC and IC sequences can be readily distinguished, in addition to AT sequences, by monitoring the sensitized emission from the anthryl probes. Thus, a comparison of the binding properties of APAC and N-Et-AMAC as well as a comparison of the binding properties of each probe as a function of DNA sequence clearly indicate the ability of the propyl side chain on the intercalator to position the hydrogen bonding group in the minor groove of the helix. Both probes can induce photochemical DNA strand damage that results in sugar-phosphate backbone cleavage. The photocleavage of the supercoiled DNA results in nicked circular DNA with little or no linear DNA suggesting that single stranded cleavage predominates. Most likely, the O2 of thymine in AT bases and O2 of cytosine in IC sequences interact with the aminopropyl side chain of APAC and this interaction provides the basis for the DNA nucleobase recognition by the small molecules.

A Maximum Entropy Analysis of Protein Orientations Using Fluorescence Polarization Data from Multiple Probes

Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a “figure of merit” is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution.