Dynamics of superhelical DNA studied by photon correlation spectroscopy

We have conducted photon correlation spectroscopy (PCS) studies on the plasmid pUC8 (2717 bp) in order to elucidate the internal dynamics of this superhelical DNA. We confirm that the first-order autocorrelation function of the scattered light from pUC8 solutions can be separated into two distinct exponential decay components, as first shown by Lewis et al. (R. Lewis, J.H. Huang and P. Pecora, Macromolecules 18 (1985) 944). A thorough analysis of the dependence on scattering vector K of the rates and amplitudes of the two components enables us to assign the slowly relaxing part to the center-of-mass diffusion of the DNA, while the faster component corresponds to rotational, bending and twisting motions of the superhelix. For larger K values the internal motions can be formally expressed in terms of an 'internal diffusion coefficient' Di, whose value of 2.0-2.5 X 10(-11) m2 s-1 is approximately equal to the translational diffusion coefficient predicted for a stiff DNA piece of the persistence length, 65 nm. Comparison of our measured Di values to those predicted from a recent theory of circular worm-like coils (K. Soda, Macromolecules 17 (1984) 2365) shows that the internal motions are faster than the theoretical values. One of the reasons for this discrepancy could be that the theory does not take into account torsional motions, which contribute significantly to the internal dynamics (J.C. Thomas, S.A. Allison, C.J. Appelof and J.M. Schurr, Biophys. Chem. 12 (1980) 177). At low K values, the fast relaxation of superhelical pUC8 is no longer proportional to K2, but reaches a constant value as K approaches zero. This behavior, not seen for the linearized DNA, can be interpreted in terms of rotational diffusion of a flexible rod-like molecule (T. Maeda and S. Fujime, Macromolecules 17 (1984) 2381) and supports an interwound rod-like structure for pUC8 DNA with an average end-to-end distance of 220 nm.     

Dynamics of F-actin and F-actin/filamin networks as studied by photon correlation spectroscopy

Photon correlation spectroscopy was used to study both F-actin and F-actin/filamin networks in solution. The measured autocorrelation functions were analyzed with the inverse Laplace transform CONTIN. The resulting frequency distributions consist of maximal five relatively narrow peaks. This rather unexpected finding disagrees with the frequency spectra calculated for an entangled rigid rod model. For this model, we expect spectra consisting of a single broad peak. Factors like flexibility, deviation from an experimental length distribution, interference with chemical reactions, and translation-rotation coupling that would influence the profile of the frequency distribution could be excluded by comparison of the data with model calculations or qualitative estimates. We conclude that our data for F-actin are consistent with the dynamics of an infinite network stable on the time scale of the observed modes. Further support for this interpretation is provided by a comparison with frequency spectra of actin/filamin networks. These appear to be very similar in shape. The main peak of these spectra that corresponds to the slow motions shifts to lower frequencies with increasing cross-link density. It appears that higher frequency modes of the gel are more efficiently damped with progressive cross-link density, resulting in a predominance of the slow motions in the spectra. This behavior has been already found in other systems and seems to be a general feature of cross-linked systems.     

The influence of temperature on the dynamics of protein precrystallization clusters,studied by photon correlation spectroscopy

Hen-egg white lysozyme was used for studying the influence of temperature on crystallization. The reaction was initiated at variable temperatures, covering the range between 5–50 °C, and was monitored with photon correlation spectroscopy. When aggregation was induced by addition of NaCl, the clusters formed exhibited diffusion limited aggregation behavior and crystals appeared in less than two days. In contrast, (NH₄)₂SO₄ induced aggregation took place mostly in the cross-over regime. In this case, solutions either remained transparent and void of crystals or formed gels within a few weeks. In both cases the kinetics could be dynamically scaled into master curves indicating that the precrystallization formed aggregates are fractals resulting from different collision processes.     

Viral DNA packaging studied by fluorescence correlation spectroscopy

The DNA packaging machinery of bacteriophage T4 was studied in vitro using fluorescence correlation spectroscopy. The ATP-dependent translocation kinetics of labeled DNA from the bulk solution, to the phage interior, was measured by monitoring the accompanied decrease in DNA diffusibility. It was found that multiple short DNA fragments (100 basepairs) can be sequentially packaged by an individual phage prohead. Fluorescence resonance energy transfer between green fluorescent protein donors within the phage interior and acceptor-labeled DNA was used to confirm DNA packaging. Without ATP, no packaging was observed, and there was no evidence of substrate association with the prohead.     

Motility evaluation of human spermatozoa by photon correlation spectroscopy.

The application of photon correlation spectroscopy for the evaluation of motility parameters of undiluted human sperm is investigated. Measurements on semen samples, selected visually as good (i.e., fraction motile spermatozoa larger than 0.6 and a positive appreciation of the motion), gave estimates of the fraction motile spermatozoa, reproducible within 10%, and of the mean velocity of the motile cells, reproducible within 5%.     

The measurement of bacterial translation by photon correlation spectroscopy.

Photon correlation spectroscopy is shown to be a practical technique for the accurate determination of translational speeds of bacteria. Though other attempts have been made to use light scattering as a probe of various aspects of bacterial motility, no other comprehensive studies to establish firmly the basic capabilities and limitations of the technique have been published. The intrinsic accuracy of the assay of translational speeds by photon correlation spectroscopy is investigated by analysis of synthetic autocorrelation data; consistently accurate estimates of the mean and second moment of the speed distribution can be calculated. Extensive analyses of experimental preparations of Salmonella typhimurium examine the possible sources of experimental difficulty with the assay. Cinematography confirms the bacterial speed estimates obtained by photon correlation techniques.     

Motility evaluation of bull spermatozoa by photon correlation spectroscopy.

Quasi-elastic light scattering techniques are employed to evaluate motility of bovine spermatozoa. The electric field correlation function CE(k, tau) has two components CE(k, tau) = alphafm+(1-alpha)fd, where fm is the correlation function due to motile cells, fd is due to dead cells and alpha is the fraction of motile cells. A function which is a linear combination of the experimentally measured fd and an empirically determined function fm is fit to the CE(k, tau) data. In this way, alpha and the approximate analytic form of fm are determined. The distribution of swimming speeds P(v) is derived from fm using an inverse Fourier sine transform procedure. Results for the time dependence of motility of bull sperm in Hank's balanced salt solution are presented.     

Characterization of molecularly imprinted polymer nanoparticles by photon correlation spectroscopy

We follow template‐binding induced aggregation of nanoparticles enantioselectively imprinted against (S)‐propranolol, and the non‐imprinted ones, using photon correlation spectroscopy (dynamic light scattering). The method requires no separation steps. We have characterized binding of (R,S)‐propranolol to the imprinted polymers and determined the degree of non‐specificity by comparing the specific binding with the results obtained using non‐imprinted nanoparticles. Using (S)‐propranolol as a template for binding to (S)‐imprinted nanoparticle, and (R)‐propranolol as a non‐specific control, we have determined range of concentrations where chiral recognition can be observed. By studying aggregation induced by three analytes related to propranolol, atenolol, betaxolol, and 1‐amino‐3‐(naphthalen‐1‐yloxy)propan‐2‐ol, we were able to determine which parts of the template are involved in the specific binding, discuss several details of specific adsorption, and the structure of the imprinted site. Copyright © 2014 John Wiley & Sons, Ltd.     

The elasticity of synthetic phospholipid vesicles obtained by photon correlation spectroscopy

Osmotic-swelling experiments were conducted on a variety of preparations of "uniform" unilamellar vesicle systems. The synthetic lipid preparations included both vesicles produced by extrusion through polycarbonate ultrafiltration membranes and vesicles produced by the pH-adjustment method. The vesicles were monitored by photon correlation spectroscopy during swelling as the osmolarity of the external solution was decreased. Contrary to our previously reported results [Aurora, T. S., Li, W., Cummins, H. Z., & Haines, T. H. (1985) Biochim. Biophys. Acta 820, 250-258; Li, W., & Haines, T. H. (1986) Biochemistry 25, 7477-7483; Li, W., Aurora, T. S., Haines, T. H., & Cummins, H. Z. (1986) Biochemistry 25, 8220-8229; Haines, T. H., Li, W., Green, M., & Cummins, H. Z. (1987) Biochemistry 26, 5439-5447] large unilamellar vesicles produced from acidic lipids by the pH-adjustment technique were highly polydisperse and did not swell in a manner that permitted the computation of a Young's modulus, presumably due to the polydispersity. Also contrary to our previous reports, membranes derived from bovine submitochondrial particles did not produce evidence of swelling when subjected to similar protocols. Analysis of osmotic swelling of extruded unilamellar vesicles has allowed us to assign Young's moduli for bilayers of dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol, in the range (5-8) x 10(8) and (3-6) x 10(8) dyn/cm2, respectively. The diameters and polydispersites obtained with electron microscopy and photon correlation spectroscopy were compared directly and with computer-modeling techniques. While excellent agreement was obtained for distributions with low polydispersity (approximately greater than 0.1), serious disagreement was found when the polydispersity exceeded approximately 0.2.     

Hindered diffusion in polymeric solutions studied by fluorescence correlation spectroscopy

Diffusion of molecules in the crowded and charged interior of the cell has long been of interest for understanding cellular processes. Here, we introduce a model system of hindered diffusion that includes both crowding and binding. In particular, we obtained the diffusivity of the positively charged protein, ribonuclease A (RNase), in solutions of dextrans of various charges (binding) and concentrations (crowding), as well as combinations of both, in a buffer of physiological ionic strength. Using fluorescence correlation spectroscopy, we observed that the diffusivity of RNase was unaffected by the presence of positively charged or neutral dextrans in the dilute regime but was affected by crowding at higher polymer concentrations. Conversely, protein diffusivity was significantly reduced by negatively charged dextrans, even at 0.4 μM (0.02% w/v) dextran. The diffusivity of RNase decreased with increasing concentrations of negative dextran, and the amount of bound RNase increased until it reached a plateau of ∼80% bound RNase. High salt concentrations were used to establish the electrostatic nature of the binding. Binding of RNase to the negatively charged dextrans was further confirmed by ultrafiltration.     

Determination of protein size in chromatography column eluants by on-line photon correlation spectroscopy

The dynamic light scattering technique of photon correlation spectroscopy has been used to determine biomacromolecule hydrodynamic radius in solutions flowing at rates similar to those experienced in liquid chromatographic separation systems. Such analyses can be performed rapidly (less than 5 s). The potential of the technique as an on-line noninvasive monitor for liquid chromatography is discussed.     

Interaction of lactoferrin with plasmid DNA studied by laser correlation spectroscopy]

The interactions of lactoferrin with plasmid DNAs substantially differing in the number of affine regions were studied. The dissociation constants for protein-nucleic acid complexes were determined. The possibility of standardizing the conditions for preparing multicomponent systems for gene-substituting therapy by laser correlation spectroscopy is discussed.     

Study of interaction of ceruloplasmin,lactoferrin, and myeloperoxidase by photon correlation spectroscopy

In this work, the diameters of protein complexes formed upon interaction of ceruloplasmin (CP) with lactoferrin (LF) and myeloperoxidase (MPO) were determined. Gage dependence of the diameter of protein particles (myoglobin, albumin, LF, CP, MPO, aldolase, ferritin) on their molecular mass logarithm was calculated. The diameter of a complex formed upon mixing CP and LF was 8.4 nm, which is in line with the radius of gyration obtained previously when the 1CP-1LF complex was studied by small-angle X-ray scattering. The diameter of a complex formed upon interaction of CP with MPO is 9.8 nm, corresponding to the stoichiometry 2CP: 1MPO. The diameter of a complex formed when LF is added to the 2CP-1MPO complex is 10.7 nm. The latter is consistent with the notion of a pentameric structure 2LF-2CP-1MPO with molecular mass of about 585 kDa.     

Determination by photon correlation spectroscopy of particle size distributions in lipid vesicle suspensions.

A method of determining particle size distributions in lipid vesicle preparations is outlined. A vesicle suspension is modeled as a polydisperse mixture of spherical shells. The distribution of particle sizes in this mixture is approximated by a continuous, piecewise linear function called a first-order spline. Excellent simultaneous fits to photon correlation spectroscopy data gathered at several different angles are presented. An error analysis is included to indicate the resolution of the method.     

Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy

Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in "off" and "on" switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypeptide chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in unfolded proteins can still proceed quite efficiently in spite of hindered diffusion caused by high macromolecular content.     

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple‐helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ～60‐80°C for hydrated samples. We report high temperature Raman spectra for freeze‐dried cartilage samples that show an increase in laser‐excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H₂O is retained within the freeze‐dried tissue. This is confirmed by thermogravimetric analysis that show 5‐7 wt% H₂O remaining within freeze‐dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re‐hydration following recovery indicate that the capacity of the denatured collagen to re‐absorb water is reduced. Our results are important for revealing the presence of bound H₂O within the collagen component of connective tissue even after freeze‐drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.     

Simultaneous measurements of sedimentation and diffusion coefficients using photon correlation spectroscopy

A simple experimental demonstration is described to show that photon correlation spectroscopy can be used to measure simultaneously the diffusion coefficient and, by the Doppler effect, the sedimentation coefficient of a small suspended particle. Calculation shows that the method should be readily extendable to particles with sedimentation coefficients of 5 S.