Our theme of measuring forces between molecules has been enlarged this year, to look down to the intimacies of specific vs. non-specific protein/DNA and up to the large-scale disturbances seen on arrays of molecules confined between walls. Our principal tool has been the application of osmotic stress, systematic variation of the activity of water to examine changes in molecular conformation and packing coupled with the physics of interaction and assembly. Our earlier studies had shown that there is more water expelled between regulatory protein and DNA making specific contact than with non-specific binding. Even one mutation in the 6 base pairs that bind the restriction endonuclease EcoRI weakens binding strength and residual intermolecular solvation to that of completely non-specific binding. However, the effect of the mutant pebble in the shoe can be mitigated by application of higher osmotic stress to reach the dehydration of specific binding. A competitive binding assay developed this year is allowing systematic investigation of the relation between specificity, binding strength and duration of molecular association. DNA-lipid condensates have been examined by small-angle x-ray diffraction to reveal many ways the negatively charged DNA can pack, particularly with positively charged lipids. Besides being a model system for molecular assembly, these condensates are used to facilitate DNA uptake in transfection. In parallel with these studies are those of DNA alone in various salt solutions where liquid-crystalline properties create various packing depending on DNA concentration and the strength of molecular interaction. These observations on DNA packing mediated by intermolecular forces are coupled with several theoretical physical formulations on the interaction between molecules not only as the intermolecular force varies with distance but also with the angle between the axes of the DNA. The advance this year was to formulate interactions that depend on the positional fluctuations of the ions that cluster around the DNA, in particular the demonstration when DNA molecules need not interact in pairs. A large study has begun, involve five different labs using the same lipid materials, built our theory of the powerful 'vapor pressure paradox'. Membranes and stiff macromolecules can pack differently when bounded by hard walls than when they are in macroscopic solutions. Disturbances created by the walls can penetrate millimeters into some samples, something to think about when the packing of DNA or membranes must sometimes take place within the micron dimensions of a virus. Related studies on the energy of packing molecules have allowed us to create 'phase diagrams' for the organization of bilayers in different multilayer arrays and to begin to measure the heats of crystallization of proteins. It is hoped that the necessary systematic studies will provide a strategy of crystallization necessary for x-ray determination of structure.
