Gotowa bibliografia na temat „Single polymer biophysics”

Thermal fluctuations of individual actin filaments confined in rectangular microchannels with dimensions similar to the mesh size of the cytoskeleton in eukaryotic cells are studied experimentally using fluorescence microscopy and theoretically by a combination of analytical methods and Monte Carlo simulations. Compared to freely fluctuating filaments, long filaments confined in narrow channels exhibit enhanced tangent correlations and a characteristic shape of their correlation function. The tangent correlation function is calculated analytically by approximating the confining geometry by a parabolic potential. This approximation is validated by Monte Carlo simulations. For the quantitative analysis of experimental data additional corrections for image analysis effects have to be included, for which we provide a modified analytical approximation formula which is corroborated by simulations. This allows us to obtain both the persistence length LP describing the bending rigidity of the polymer and the deflection length λ characterizing confinement effects from fits to the experimental data. Our results confirm the scaling relation λ ∝ d2/3 between the deflection length and the channel width d.

In this thesis, single-molecule experiments using LOT are employed to extract accurate information about the thermodynamics and kinetics of various molecular systems, with special emphasis on the elastic properties of single-stranded DNA (ssDNA). The thesis is divided in three parts. Part I provides a general description of the research field as well as the main theoretical framework for the basic concepts that will be developed in parts II and III. In Chapter 2 the miniTweezers and the experimental setup used throughout the thesis is described, as well as the physical basis of its working mechanisms, introducing the phenomenon of optical trapping. Chapter 3 contains a brief introduction of the biomolecules of study in this thesis, with an explanation of their historical discoveries, as well as their structure and function. The main focus of this chapter is on ssDNA, which is the main object of study of the thesis. Chapter 4 introduces the polymer models that are widely used in describing the elasticity of nucleic acids and proteins. Specifically, the Freely-Jointed Chain and Worm-Like Chain models are presented. Part II deals with the elasticity of single-stranded DNA. This is the main part of the thesis, and it includes chapters 5-7. Chapter 5 is about the study of the elasticity of ideal ssDNA chain, i.e. the one that can be modelled as ideal polymers (presented in Chapter 4). The study of the elasticity of different DNA sequences is presented. The blocking-splint oligo technique is described, a experimental technique developed for studying the elasticity of short (tens of bases) DNA molecules. This study shows the need of using extensible models to succesfully describe ssDNA elasticity over a large range of forces, which explains the previous discrepancies on the elastic parameters obtained in different studies. We also provide an explanation for the required extensibility of the model: a transition experienced at the nucleotide level: a change in DNA sugar pucker conformation. A simple two-states model is introduced and preeliminary results regarding its energetics are presented. The characterization of the ssDNA elasticity is central for the works developed in the following chapters. Chapter 6 studies the stacking-unstacking transition for ssDNA, previously observed for certain sequences (mainly purine-rich ones). Several molecules, with different degrees of stacking, are studied by obtaining their force extension curves (FECs). A cooperative helix-coil model including heterogeneity is developed and used to fit the obtained FECs, allowing to obtain elastic parameters to describe the stacked chain. The salt dependence of the unstacking transition is also measured by studying two of the sequences by varying the salt concentration over two decades. The free energy of formation of dsDNA duplexes depends on the salt concentration. The obtained salt dependence on the stacking free-energy of ssDNA provides a possible explanation for the salt dependence of duplex formation. Chapter 7 deals with the non-specific structures that arise at low forces and high salt concentration when pulling ssDNA molecules longer than $\sim 100$ bases. A helix-coil model with cooperativity is proposed and used to extract some mean-field characteristics of these structures. 8 different sequences are studied, characterizing their elasticity and deviation from the ideal elastic behaviour. The results for a $14$kb molecule for 3 decades of varying \ce{NaCl} and \ce{MgCl2} are also shown. All experimental FECs are fitted to the helix-coil model. The model can be used to predict the formation of secondary structures at zero force. A comparison between the predicted structures from the model and those obtained from Mfold is also investigated. Part III contains two studies which also need of the correct determination of ssDNA elasticity. In Chapter 8, we study the interaction between the RecQ helicase from E. coli and DNA, i.e. how the RecQ unwinds double-stranded DNA molecules, releasing single-stranded DNA. We obtain some of its kinematic properties as well as study the entropy production of the system using the Fluctuation Theorem. In Chapter 9, the effect of DNA mismatches, i.e. non complementary base pairing, on the stability of DNA is studied. To do so, two types of experiments on several DNA sequences are performed: stretching and releasing the molecule by moving the optical trap (pulling experiments) and monitoring the folding/unfolding of the molecule passively (hopping experiments).
En aquesta tesi hem realitzat experiments fent servir pinces òptiques per tal d’extreure informació precisa sobre les propietats termodinàmiques i cinètiques de diferents sistemes moleculars, posant especial èmfasi en les propietats elàstiques de la cadena simple d’ADN (ssDNA, pel seu acrònim en anglès). La tesi es troba dividida en tres parts. A la primera part s’introdueix de forma general el camp de recerca dels experiments de molècula única, així com s’expliquen els conceptes més bàsics que es desenvoluparan en les parts II i III. La configuració experimental emprada al llarg de tota la tesi, les pinces òptiques, s’introdueix al capítol 2. Per a fer-ho, s’expliquen els principis físics de funcionament de les pinces, que es basen en l’atrapament òptic. Breument, la focalització d’un feix de llum d’alta intensitat permet atrapar i exercir forces en micropartícules dielèctriques (pilotes fetes de plàstic de la mida d’un bacteri), que són recobertes químicament de manera que la molècula d’estudi pot estirar-se, de forma individual, repetides vegades. El capítol 3 conté una breu introducció a les biomolècules que apareixen en aquesta tesi, amb una breu explicació de la seva descoberta, així com la seva estructura i funció (íntimament relacionades). Ens centrem en la descripció de la ssDNA que és el principal objecte d’estudi de la tesi. Al capítol 4 s’introdueixen els models de polímers que s’empren habitualment per a descriure l’elasticitat d’àcids nucleics i proteïnes. En concret, es descriuen els models de la Freely-Jointed Chain i la Worm-Like Chain. La Part II tracta de l’elasticitat de la ssDNA, i inclou els capítols 5, 6 i 7. El capítol 5 es basa en la caracterització de l’elasticitat de la cadena ideal de ssDNA, és a dir, aquella que pot ser modelitzada pels polímers ideals introduïts en el capítol 4. S’estudia l’elasticitat de diferents seqüències de ssDNA, introduint un nou mètode experimental, blocking-splint oligo, per tal d’ampliar el rang de forces estudiat habitualment en molècules curtes (d’una longitud de desenes de bases) de ssDNA. L’estudi mostra la necessitat d’emprar models elàstics extensibles per a la correcte caracterització de l’elasticitat de ssDNA, que explica les discrepàncies existents entre els paràmetres elàstics trobats a la literatura. També hipotetitzem que l’extensibilitat del model pot ser explicada gràcies a la transició experimentada a nivell de nucleòtids: el canvi que experimenta la distància interfosfat de l’ADN es veu modificada segons quina sigui la configuració de l’anell de desoxiribosa. Tot i que és un fenomen molt més conegut en la cadena doble d’ADN, l’apilament-desapilament de bases també s’ha observat en certes seqüències de ssDNA (especialment les que són riques en contingut de purines). Al capítol 6 s’estudien quatre molècules amb un grau d’apilament diferent a partir de les seves corbes força-extensió (FECs). Es desenvolupa un model helix-coil (hèlix-cabdell) per tal d’ajustar les FECs, fet que permet d’obtenir, indirectament, les propietats elàstiques de la cadena apilada. També s’estudia la dependència d’aquesta transició variant la concentració de sal dels experiments en més de dos ordres de magnitud. A través d’aquests experiments, trobem una dependència amb la concentració de sal de l’energia lliure de formació de l’apilament de la ssDNA, fet que ens permet explicar, parcialment, la dependència que es troba en la literatura per la hibridació de la cadena doble d’ADN. El capítol 7 tracta de la formació d’estructures no específiques que apareixen a forces baixes i a concentració de sal alta per a molècules de ssDNA de més de ~100bases. Es proposa un model helix-coil amb cooperativitat per tal de caracteritzar propietats de camp mitjà de les estructures estudiades. S’estudien vuit seqüències diferents, entre 120 i ~14000 bases, i es caracteritza el seu desviament respecte de la corba elàstica ideal amb el model. També s’estudia la dependència de l’estructura secundària de la ssDNA en funció de la concentració de la sal. Analitzant experiments variant la concentració de MgCl2 i NaCl, aconseguim reproduir les FECs a partir de fer dependre els paràmetres del model amb la sal. Finalment, el model desenvolupat ens permet predir la formació d’estructura secundària a força zero (fet que no podem detectar directament a partir d’experiments d’espectroscopia de forces). Es comparen les previsions del model amb les trobades per Mfold, trobant una compatibilitat per als resultats per a molècules de de menys de 1000 bases. La darrera part se centra en col·laboracions que he fet durant a tesi i que necessiten una determinació precisa de les propietats elàstiques de la ssDNA. Al capítol 8 s’estudia la interacció entre l’helicasa del bacteri E. coli i l’ADN, que s’encarrega d’obrir la cadena doble d’ADN, alliberant ssDNA. S’extreuen les seves propietats cinètiques, com la velocitat de translocació – obtenim, independentment de la força aplicada, d’uns 50bp/s, d’acord amb la literatura –. També n’estudiem les seves propietats termodinàmiques, a partir del Teorema de Fluctuació. Finalment, al capítol 9 s’estudien els efectes de certs defectes en molècules d’ADN. A partir d’experiments fora de l’equilibri s’extrau la penalització que suposa per a la hibridització d’ADN la presència d’aquestes bases no complementàries (és a dir, que no són enllaços de A-T o G-C).

Diese Dissertation konzentriert sich auf die Untersuchung multivalenter Wechselwirkungen zwischen Hämagglutinin (HA) sowie Neuraminidase (NA) zweier Stämme des Influenzavirus (H1N1 und H3N2) und dem zellulären Liganden Sialinsäure (SA) unter Verwendung von Rasterkraftmikroskopie und Einzelmolekülkraftspektroskopie (SMFS). Bindungskräfte sowie Dissoziations- und Assoziationskinetiken, zusammen mit den intermolekularen Potentiallandschaften wurden, nach bestem Wissen erstmalig, auf Einzelmolekülebene mittels SMFS quantifiziert. Zu diesem Zweck wurden mono- und multivalente SA-Liganden (SAPEGLA und dPGSA) eingesetzt. Abweichungen der experimentellen Kraftspektren vom klassischen Kramers-Bell-Evans-Modell vorhergesagten Verhalten wurden durch das Friddle-Noy-De Yoreo-Model berücksichtigt. NA beider Virusstämme zeigte trotz ähnlicher Bindungskräfte eine stabilere Bindung mit SA als HA und dissoziierte 3 – 7 mal langsamer. Es wird vermutet, dass die höhere Stabilität die geringere Oberflächendichte von NA auf der Virushülle im Vergleich zu HA ausgleicht. Die Bindungskräfte eines SAPEGLA-Clusters nehmen mit der Anzahl der Bindungen und die Dissoziationskinetik folgt dem theoretisch vorhergesagten Trend. Die Dissoziationsrate von NA ist etwa 6-mal höher ist als ihre katalytische Rate, weshalb Mehrfachbindungen zur Spaltung von SA erforderlich sind. Die Dissoziationsrate von N1 in der gleichen Größenordnung wie die von H3 und es wird vermutet, dass derartige Ähnlichkeiten die Übertragbarkeit des Virus begünstigen. Darüber hinaus wird gezeigt, dass die thermische Stabilität von HA-dPGSA höher ist als von HA-SAPEGLA und im Bereich von 3 - 4 Einzelbindungen liegt, was für NA-dPGSA nicht beobachtet werden konnte. Daher bindet dPGSA spezifisch und kooperativ multivalent an HA. Kompetitive Bindungstests zeigen, dass SMFS zum Screening von antiviralen Inhibitoren verwendet werden und Zugang zu deren Design auf Einzelmolekülebene liefern könnte.
This thesis focuses on studying multivalent interactions between influenza virus hemagglutinin (HA) as well as neuraminidase (NA) of two viral strains (H1N1 and H3N2) and the cellular ligand sialic acid (SA) by using scanning force microscopy and single molecule force spectroscopy (SMFS). Unbinding forces as well as dissociation and association kinetics together with the free energy landscapes were, to the best knowledge for the first time, individually quantified on the single molecule level using SMFS. To this extent, designed synthetic monovalent (SAPEGLA) and multivalent (dPGSA) SA displaying ligands were employed. Surprisingly, the experimental force spectra did not show the log-linear trend predicted by the classical Kramers-Bell-Evans model, but rather follow the more recent Friddle-Noy-De Yoreo model. NA of both viral strains forms a more stable bond with SA than HA, and dissociates 3 to 7 times slower. It is reasoned that the higher stability compensates for the lesser amount of NA compared to HA that is typically found on the viral envelope. The unbinding forces of the cluster of SAPEGLA increased gradually with the number of bonds in the cluster and the dissociation kinetics follow the theoretically predicted trend. The dissociation rate of NA was found to be about 6 times higher than its catalytic rate, indicating that multiple bonds are needed for cleavage of SA. The dissociation rate of N1 is on the same order as that of H3, suggesting that these similarities between the two strains favor transmissibility. The thermal stability of the HA-dPGSA bond is higher than the HA-SAPEGLA reaching that of three to four single bonds, proving specificity and cooperativity. Such an enhancement could not be observed for the binding of NA. This thesis also shows that SMFS could be used as a tool to screen antiviral inhibitors in competitive binding assays, which may contribute insight into the design of antiviral inhibitors on the single molecule level.

In this thesis, the response of DNA and RNA to linear and torsional mechanical stress is studied using coarse-grained models. Inspired by single-molecule assays developed over the last two decades, the end-to-end extension, buckling and torque response behaviour of the stressed molecules is probed under conditions similar to experimentally used setups. Direct comparison with experimental data yields excellent agreement for many conditions. Results from coarse-grained simulations are also compared to the predictions of continuum models of linear polymer elasticity. A state diagram for supercoiled DNA as a function of twist and tension is determined. A novel confomational state of mechanically stressed DNA is proposed, consisting of a plectonemic structure with a denaturation bubble localized in its end-loop. The interconversion between this novel state and other, known structural motifs of supercoiled DNA is studied in detail. In particular, the influence of sequence properties on the novel state is investigated. Several possible implications for supercoiled DNA structures in vivo are discussed. Furthermore, the dynamical consequences of coupled denaturation and writhing are studied, and used to explain observations from recent single molecule experiments of DNA strand dynamics. Finally, the denaturation behaviour, topology and dynamics of short DNA minicircles is studies using coarse-grained simulations. Long-range interactions in the denaturation behaviour of the system are observed. These are induced by the topology of the system, and are consistent with results from recent molecular imaging studies. The results from coarse-grained simulations are related to modelling of the same system in all-atom simulations and a local denaturation model of DNA, yielding insight into the applicability of these different modelling approaches to study different processes in nucleic acids.

This thesis presents an analysis of the material properties and phase behavior of divalent ionic surfactant salts, and protein and polymer glasses. There has been extensive interest in understanding the phase behavior of divalent ionic surfactants due to the many applications of ionic surfactants in which they come into contact with divalent ions, such as detergency, oil recovery, and surfactant separation processes. One goal of determining the phase boundaries was to explore the option of incorporating a hydrophobic molecule into the solid phase through the micelle-to-crystal bilayer transition, either for drug delivery applications (with a biologically compatible surfactant) or for the purpose of studying the hydrophobic molecule itself. The liquid micellar and solid crystal phases of the alkaline earth metal dodecyl sulfates were investigated using calorimetry, visual inspection, solubilization of a fluorescent probe, and x-ray diffraction. The Krafft temperature and dissolution enthalpy were determined for each surfactant, and partial composition-temperature phase diagrams of magnesium dodecyl sulfate-water, calcium dodecyl sulfate-water, as well as sodium dodecyl sulfate with MgCl₂ and CaCl₂ are presented. As a proof of concept, fluorescence microscopy images showed that it is, in fact, possible to incorporate a small hydrophobic molecule, diphenylhexatriene, into the solid phase.

The second, and main, part of this thesis expands on work done previously in the lab by using the micropipette technique to study two-phase microsystems. These microsystems consist of a liquid droplet suspended in a second, immiscible liquid medium, and can serve as direct single-particle studies of drug delivery systems that are formed using solvent extraction (e.g., protein encapsulated in a biodegradable polymer), and as model systems with which to study the materials and principles that govern particle formation. The assumptions of the Epstein-Plesset model, which predicts the rate of droplet dissolution, are examined in the context of the micropipette technique. A modification to the model is presented that accounts for the effect a solute has on the dissolution rate. The modification is based on the assumption that the droplet interface is in local thermodynamic equilibrium, and that the water activity in a solution droplet can be used to determine its dissolution (or dehydration) rate. The model successfully predicts the dissolution rates of NaCl solutions into octanol and butyl acetate up to the point of NaCl crystallization. The dehydration of protein solutions (lysozyme or bovine serum albumin) results in glassified microbeads with less than a monolayer of water coverage per protein molecule, which can be controlled by the water activity of the surrounding organic medium. The kinetics of dehydration match the prediction of the activity-based model, and it is shown how the micropipette technique can be used to study the effect of dissolution rate on final particle morphology. By using a stable protein with a simple geometry (lyosyzme), this technique was be used to determine the distance dependence of protein-protein interactions in the range of 2-25 Å, providing the first calculation of the hydration pressure decay length for globular proteins. The distance-dependence of the interaction potential at distances less than 9 Å was found to have a decay length of 1.7 Å, which is consistent with the known decay length of hydration pressure between other biological materials. Biodegradable polyesters, such as poly(lactide-co-glycolide) (PLGA), are some of the most common materials used for the encapsulation of therapeutics in microspheres for long-term drug release. Since they degrade by hydrolysis, release rates depend on water uptake, which can be affected by processing parameters and the material properties of the encapsulated drug. The micropipette technique allows observations not possible on any bulk preparation method. Single-particle observations of microsphere formation (organic solvent extraction into a surrounding aqueous phase) show that as solvent leaves the microsphere and the water concentration in the polymer matrix becomes supersaturated, water phase separates and inclusions initially grow quickly. Once the concentration in the polymer matrix equilibrates with the surrounding aqueous medium, the water inclusions continue to grow due to dissolved impurities, solvent, and/or water-soluble polymer fragments resulting from hydrolysis, all of which locally lower the water activity in the inclusion. Experiments are also presented in which glassified protein microbeads were suspended in PLGA solution prior to forming the single microspheres. This technique allowed the concentration of protein in a single microbead/inclusion to be determined as a function of time.

Dissertation

Indiana University-Purdue University Indianapolis (IUPUI)
Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.

Pressure-tuning spectroscopy is a powerful tool for investigating molecular interactions. These interactions may involve organic or inorganic materials in liquid, polymeric, or crystalline media. In this article we confine our attention to organic molecules, largely in dilute solution in polymers or liquids. We demonstrate the use of high-pressure luminescence to study the effect of the environment on π* →π, π* →n and charge-transfer excitations, as well as the interaction between singlet and triplet states. In addition, we provide tests of the energy gap law for non-radiative dissipation of excitation, the role of viscosity in luminescent efficiency, and the internal consistency of various means of predicting and correlating energy transfer. Over the past 40 years, it has been amply demonstrated that high pressure is a powerful tool for studying electronic phenomena in condensed phases. The basic concept is as follows. The optical, electrical, magnetic, and chemical properties—collectively the electronic properties—of condensed phases depend on the interactions of the outer electrons on the atoms, molecules, or ions that make up the phase. Different kinds of electronic orbitals have different spatial characteristics—different radial extent, different shape (orbital angular momentum), and different diffuseness; therefore, pressure perturbs the energies associated with these orbitals in different degrees. This relative perturbation we call “pressure tuning,” and the measurement and explanation of the tuning is “pressure-tuning spectroscopy.” Pressure-tuning spectroscopy of the vibrational and rotational excitations of atoms in molecular and in crystal lattices is also an active and important field, but in this article we arc concerned mainly with electronic phenomena. We further limit this discussion primarily to organic molecules in solid polymers or liquid solutions, as these have the greatest relevance to biologically active systems. A variety of probes are used for studying electronic phenomena under high pressure, but the emphasis here is on luminescence. The presentation consists of a series of examples of various types of excitations on interactions where high pressure has been an effective tool. Only references directly relevant to each example are included. Two general references to pressure studies of molecular luminescence have been published (Drickamer, 1982, 1990).

The effect of pressure on the helix-coil transition temperature (Tm) is reported for the double-stranded polymers poly(dA)poly(dT), poly[d(A-T)], poly[d(l-C], and poly[d(G-C] and triple-stranded poly(dA)2poly(dT). The Tm increases as a function of pressure, implying a positive volume change for the transition and leading to the conclusion that the molar volume of the coil form is larger than the molar volume of the helix. From the change in Tm as a function of pressure, molar volume changes of the transition (ΔVt) are calculated using the Clapeyron equation and calorimetrically determined enthalpies. For the doublestranded polymers, ΔVt, increases in the order poly[d(l-C] < polyt[d(A-T)] < poly(dA)-poly(dT) < polylcl(G-C)]. The value of ΔVt, for the triple-stranded to single-stranded transition of poly(dA) 2poly(dT) is larger than that of poly[d(G-C)I. The magnitude of ΔVt increases with salt concentration in all cases studied; however, the change of ΔVt with salt concentration depends on the sequence of the DNA and the number of strands involved in the transition. In the model proposed to explain the results, the overall molar volume change of the transition is a function of a negative volume change arising from changes in the electrostatic interactions of the DNA strands, and a positive volume change due to unstacking the bases. The model predicted the direction of the change in the ΔVt for several experiments. The magnitude of AVJ increases with counter ion radius, thus for polyld(A-T)], ΔVt, increases in the series Na+ , K+, Cs+, The ΔVt also increases if the charge on the phosphodiester groups is removed. The kinetics of the formation of double-stranded (dA)19(dT)19 in 50 mM NaCI are slowed approximately 14-fold at 200 MPa relative to atmospheric pressure. The implied volume of activation of +37 ml mol−l in the direction of this change is also in agreement with the proposed model. The stability of double- and triple-stranded DNA helices in water around neutral pH depends on the base composition and sequence, as well as on the ionic strength of the solution. Each of these dependencies also defines how DNA interacts with water.