Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer.

Many studies of specific protein-nucleic acid binding use short oligonucleotides or restriction fragments, in part to minimize the potential for nonspecific binding of the protein. However, when the specificity ratio is low, multiple nonspecifically bound proteins may occupy the region of DNA corresponding to one specific site; this situation was encountered in our recent calorimetric study of binding of integration host factor (IHF) protein to its specific 34-bp H' DNA site. Here, beginning from the analytical McGhee and von Hippel infinite-lattice nonspecific binding isotherm, we derive a novel analytic isotherm for nonspecific binding of a ligand to a finite lattice. This isotherm is an excellent approximation to the exact factorial-based Epstein finite lattice isotherm even for short lattices and therefore is of great practical significance for analysis of experimental data and for analytic theory. Using this isotherm, we develop an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecific binding sites. Analysis of calorimetric data for IHF-H' DNA binding using this treatment yields enthalpies and binding constants for both specific and nonspecific binding and the nonspecific site size. This novel analysis demonstrates the potential contribution of nonspecific binding to the observed thermodynamics of specific binding, even with very short DNA oligomers, and the need for reverse (constant protein) titrations or titrations with nonspecific DNA to resolve specific and nonspecific contributions. The competition treatment is useful in analyzing low-specificity systems, including those where specificity is weakened by mutations or the absence of specificity factors.

Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding.

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H' site from lambda-phage DNA. At low to moderate [K(+)] (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (approximately 10(2)) and a very small non-specific site size. In this [K(+)] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K(+)]. Above 100 mM K(+), only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K(+)]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 A of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K(+)]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K(+)] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.

Enthalpy and heat capacity changes for formation of an oligomeric DNA duplex: interpretation in terms of coupled processes of formation and association of single-stranded helices.

The thermodynamics of self-assembly of a 14 base pair DNA double helix from complementary strands have been investigated by titration (ITC) and differential scanning (DSC) calorimetry, in conjunction with van't Hoff analysis of UV thermal scans of individual strands. These studies demonstrate that thermodynamic characterization of the temperature-dependent contributions of coupled conformational equilibria in the individual "denatured" strands and in the duplex is essential to understand the origins of duplex stability and to derive stability prediction schemes of general applicability. ITC studies of strand association at 293 K and 120 mM Na+ yield an enthalpy change of -73 +/- 2 kcal (mol of duplex)-1. ITC studies between 282 and 312 K at 20, 50, and 120 mM Na+ show that the enthalpy of duplex formation is only weakly salt concentration-dependent but is very strongly temperature-dependent, decreasing approximately linearly with increasing temperature with a heat capacity change (282-312 K) of -1.3 +/- 0.1 kcal K-1 (mol of duplex)-1. From DSC denaturation studies in 120 mM Na+, we obtain an enthalpy of duplex formation of -120 +/- 5 kcal (mol of duplex)-1 and an estimate of the corresponding heat capacity change of -0.8 +/- 0.4 kcal K-1 (mol of duplex)-1 at the Tm of 339 K. van't Hoff analysis of UV thermal scans on the individual strands indicates that single helix formation is noncooperative with a temperature-independent enthalpy change of -5.5 +/- 0.5 kcal at 120 mM Na+. From these observed enthalpy and heat capacity changes, we obtain the corresponding thermodynamic quantities for two fundamental processes: (i) formation of single helices from disordered strands, involving only intrastrand (vertical) interactions between neighboring bases; and (ii) formation of double helices by association (docking) of single helical strands, involving interstrand (horizontal and vertical) interactions. At 293 K and 120 mM Na+, we calculate that the enthalpy change for association of single helical strands is approximately -64 kcal (mol of duplex)-1 as compared to -210 kcal (mol of duplex)-1 calculated for duplex formation from completely unstructured single strands and to the experimental ITC value of -73 kcal (mol of duplex)-1. The intrinsic heat capacity change for association of single helical strands to form the duplex is found to be small and positive [ approximately 0.1 kcal K-1 (mol of duplex)-1], in agreement with the result of a surface area analysis, which also predicts an undetectably small heat capacity change for single helix formation.

SECReT of the eukaryotic large ribosomal subunit.

The large ribosomal subunit of the thermophilic fungus Thermomyces lanuginosus was reconstructed in three dimensions from electron micrographs. This is the first reported reconstruction of the eukaryotic complex and demonstrates specific structural features such as an intersubunit canyon and a potential channel for the nascent peptide chain.

Computer applications in quality assurance.

The computer is rapidly becoming an essential tool for the physician. Proper use of computers in practice will help physicians achieve both higher levels of quality and greater consistency in patient care. Only with computers can physicians rapidly access and process all the data now needed to best address the needs of their patients. As computer use in practice becomes the standard, the inability to use these tools will be incompatible with quality care. The computer is rapidly becoming essential to modern medical management strategies that demand efficiency, accuracy, and cost effectiveness in response to patient demands for assurance that quality care is being delivered.

Generating medical documentation through voice input: the emergency room.

We look in one to three years to continuous speech recognition without sacrificing large vocabulary recognition or speaker independence. At that point we will lose some of the risk management value of structured reporting. But if the experience of emergency medicine is any indicator, it is clear that ASR is here to stay, and will increasingly become the gateway to the hospital information systems of the 90s.