Kenneth J. Breslauer

Hydration and partial compressibility of biological compounds

We review the results of compressibility studies on proteins, nucleic acids, and systematically altered low molecular weight compounds that model the constituents of these biopolymers. The model compound data allow one to define the compressibility properties of water surrounding charged, polar, and nonpolar groups. These results, in conjunction with compressibility data on proteins and nucleic acids, were used to define the properties of water that is perturbed by the presence of these biopolymers in aqueous solutions. Throughout this review, we emphasize the importance of compressibility data for characterizing the hydration properties of solutes (particularly, proteins, nucleic acids, and their constituents), while describing how such data can be interpreted to gain insight into role that hydration can play in modulating the stability of and recognition between biologically important compounds.

Calorimetry : a tool for DNA and ligand-DNA studies

Publisher Summary This chapter discusses the unique information that can be obtained by the application of selected calorimetric techniques to the study of DNA. The chapter demonstrates calorimetry to be a powerful tool in biophysical research, capable of uniquely providing thermodynamic and extrathermodynamic characterizations of DNA structure, conformational transitions, and ligand interactions. A differential scanning calorimeter is an instrument that allows to measure continuously the heat capacity of a system as a function of temperature. Isothermal mixing calorimetry has been used in nucleic acid studies to characterize the influence of metal ion binding; to determine the enthalpy of duplex formation from the mixing of two complementary strands; to measure the energetics of pH-induced changes in nucleic acid structure; and to determine the enthalpy of small ligand-DNA interactions and protein–DNA interactions. Multifrequency calorimetry has been applied to the analysis of membrane phase transitions and to the folding–unfolding transition of the protein cytochrome c .

Thermodynamic analysis of biomolecules: a volumetric approach

Fundamental thermodynamic relationships reveal that volumetric studies on molecules of interest can yield useful new information. In particular, appropriately designed volumetric studies can characterize the properties of molecules as a function of solution conditions, including the role of solvation. Until recently, such studies on biologically interesting molecules have been limited because of the lack of readily available instrumentation with the requisite sensitivity; however, during the past decade, advances in the development of highly sensitive, small-volume densimetric, acoustic and high-pressure spectroscopic instrumentation have enabled biological molecules to be subjected to a wide range of volumetric studies. In fact, the volumetric methods used in these studies have already provided unique insights into the molecular origins of the intramolecular and intermolecular recognition events that modulate biomolecular processes. Of particular note are recent volumetric studies on globular proteins and nucleic acid duplexes. These studies have provided unique insights into the role of hydration in modulating the stabilities of these biopolymers, as well as their conformational transitions and ligand-binding properties.

The thermodynamics of DNA structures that contain lesions of guanine tetrads

It is becoming increasingly apparent that energetic as well as structural information is required to develop a complete appreciation of the critical interrelationships between structure, energetics, and biological function. Motivated by this recognition, we have reviewed in this article the current state of the thermodynamic databases associated with lesion-containing DNA duplexes and DNA quadruplexes, while highlighting important considerations concerning the methods used to obtain the requisite data.

Effect of sucrose on the structure, mechanical strength and thermal properties of corn extrudates

Abstract The effects of sucrose on extrusion process parameters and the structural, mechanical and thermal characteristics of the resultant extrudates have been investigated. To this end, sucrose in concentrations between 0 and 10% by weight was added to corn meal prior to extrusion at two levels of moisture (15 and 20%). The resulting data revealed the following significant features. (i) At the higher moisture level, sucrose progressively reduced the specific mechanical energy, while exhibiting little effect at the lower moisture level. (ii) At both moisture levels, sucrose increased the bulk density and reduced the cell size; this effect was progressive for high extrusion moisture samples and evident only at high sucrose contents for low extrusion moisture samples. (iii) In extrudate samples equilibrated to moisture contents between 12 and 17% wt, sucrose progressively plasticized the structures, as assessed by compression, dynamic mechanical spectrometry, and differential scanning calorimetry. In the aggregate, these results showed that the addition of sucrose requires a modification of the extrusion operating conditions to produce extrudates with optimal textural and storage properties.

Calorimetry of proteins and nucleic acids

The availability of sensitive calorimetric instrumentation has led to a considerable increase in thermodynamic studies of proteins, nucleic acids, and their interactions. This article reviews some of the recent contributions of calorimetry to characterizing the thermodynamic origins of protein and nucleic acid stability and conformational preferences, as well as the interactions of proteins with each other, with small molecules, and with nucleic acids.

Thermodynamics of drug-DNA interactions.

Batch calorimetry, differential scanning calorimetry (DSC), uv/vis absorption spectroscopy, fluorescence spectroscopy, and circular dichroism (CD), have been used to detect, monitor, and thermodynamically characterize the binding of daunomycin, dipyrandenium, dipyrandium, and netropsin to poly d(AT) and actinomycin D to salmon testes (ST) DNA. The following thermodynamic binding profiles have been obtained. (table; see text) All the poly d(AT) binding studies were done at 25 degrees C while actinomycin binding to ST DNA was performed at 1 degree C to enhance drug solubility. These thermodynamic parameters are interpreted in terms of specific interactions that have been proposed as part of models for the binding of each drug.

The thermodynamics of drug-DNA interactions: ethidium bromide and propidium iodide.

We report the first calorimetrically-derived characterization of the thermodynamics of ethidium bromide (EB) and propidium iodide (PI) binding to a series of nucleic acid host duplexes. Our spectroscopic and calorimetric measurements yield the following results: 1) At low salt (16mM Na+) and 25 degrees C. PI binds more strongly than EB to a given host duplex. The magnitude of this PI preference depends only marginally on base sequence, with AT base pairs showing a greater PI preference than GC base pairs. 2) The enhanced binding of PI relative to EB at low salt and 25 degrees C reflects a more favorable entropic driving force for PI binding. 3) The PI binding preference diminishes at higher salt concentrations (216mM). In other words, the binding preference is electrostatic in origin. 4) The salt dependence of the binding constants (delta lnKb/delta ln[Na+]) reveal that PI binds as a dication while EB binds as a monocation. 5) PI and EB both exhibit impressive enthalpy-entropy compensations when they bind to the deoxy homopolymers poly dA.poly dT and poly dA.poly dU. We have observed a similar enthalpy-entropy compensation for netropsin binding to the poly dA.poly dT homopolymer duplex. We therefore conclude that the compensation phenomenon is an intrinsic property of the host duplex rather than reflecting a property of the binding ligand. 6) When either PI or EB bind to the corresponding ribo homopolymer (poly rA.poy rU) we do not observe the enthalpy-entropy compensation that characterizes the binding to the deoxy homopolymer. 7) EB and PI both bind more strongly to poly d(AT).poly d(AT) than to poly d(AU).poly d(AU). Specifically, the absence of the thymine methyl group in poly d(AU).poly d(AU) reduces the binding constant of both drugs by a factor of four. This reduction in binding is due to a less favorable entropy change. In this paper we present and discuss possible molecular origins for our observed thermodynamic and extra-thermodynamic data. In particular, we evoke solvent effects involving both the drugs and the host duplexes when we propose molecular interpretations which are consistent with our thermodynamic data.

Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: implications for triplet expansion diseases.

We have embedded the hexameric triplet repeats (CAG)6 and (CTG)6 between two (GC)3 domains to produce two 30-mer hairpins with the sequences d[(GC)3(CAG)6(GC)3] and d[(GC)3(CTG)6(GC)3]. This construct reduces the conformational space available to these repetitive DNA sequences. We find that the (CAG)6 and (CTG)6 repeats form stable, ordered, single-stranded structures. These structures are stabilized at 62°C by an average enthalpy per base of 1.38 kcal·mol−1 for the CAG triplet and 2.87 kcal·mol−1 for the CTG triplet, while being entropically destabilized by 3.50 cal·K−1·mol−1 for the CAG triplet and 7.6 cal·K−1·mol−1 for the CTG triplet. Remarkably, these values correspond, respectively, to 1/3 (for CAG) and 2/3 (for CTG) of the enthalpy and entropy per base values associated with Watson–Crick base pairs. We show that the presence of the loop structure kinetically inhibits duplex formation from the two complementary 30-mer hairpins, even though the duplex is the thermodynamically more stable state. Duplex formation, however, does occur at elevated temperatures. We propose that this thermally induced formation of a more stable duplex results from thermal disruption of the single-stranded order, thereby allowing the complementary domains to associate (perhaps via “kissing hairpins”). Our melting profiles show that, once duplex formation has occurred, the hairpin intermediate state cannot be reformed, consistent with our interpretation of kinetically trapped hairpin structures. The duplex formed by the two complementary oligonucleotides does not have any unusual optical or thermodynamic properties. By contrast, the very stable structures formed by the individual single-stranded triplet repeat sequences are thermally and thermodynamically unusual. We discuss this stable, triplet repeat, single-stranded structure and its interconversion with duplex in terms of triplet expansion diseases.

Spectroscopic and volumetric investigation of cytochrome c unfolding at alkaline pH: characterization of the base-induced unfolded state at 25 degrees C.

We have measured at 25°C the relative specific sound velocity increment, [u], and the partial specific volume, v°, of cytochrome c as a function of pH. Our data reveal that the base‐induced native to unfolded transition of the protein is accompanied by a volume decrease of 0.014 cm3 g−1 and a compressibility decrease of 3.8 × 10−6 cm3 g−1 bar−1. These results allow us to conclude that, relative to a fully unfolded conformation, the base‐denatured state of cytochrome c has only 70 to 80% of its surface area exposed to the solvent. Recently, we reported a similar result for the acid‐denatured state of cytochrome c. Thus, insofar as solvent exposure is concerned, both the base‐ and the acid‐induced unfolded states of cytochrome c retain some order, with 20 to 30% of their surface areas remaining solvent‐inaccessible. We discuss the implications of this result in terms of defining potential intermediate states in protein folding pathways.—Chalikian, T. V., Gin‐ dikin, V. S., Breslauer, K. J. Spectroscopic and volumetric investigation of cytochrome c unfolding at alkaline pH: characterization of the base‐induced unfolded state at 25°C. FASEB J. 10, 164‐170 (1996)