The ability to measure directly forces between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, the last 1-1.5 nanometers separation. The universality of the force characteristics observed for a wide variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, has led us to conclude that the energy associated with changes in structuring water between surfaces dominates intermolecular forces. ? Exclusion of solutes from macromolecular surfaces: The stability and dynamics of biomacromolecules are greatly affected by their interaction with small solutes. Our results indicate that solute exclusion is due to repulsive hydration forces. We have now examined several osmolytes commonly used to either stabilize or disrupt protein native structure. We are particularly interested in their interaction with hydrophobic groups. The current debate centers on the relative contributions of osmolyte interactions with the peptide backbone and with the hydrophobic core of proteins. Using the osmotic stress technique and x-ray scattering we are able to measure the exclusion of solutes from ordered arrays of the hydrophobically modified polysaccharide hydroxypropylcellulose (HPC) as a function of the distance between HPC polymers. In agreement with other measurements, we see little interaction of urea with HPC. Protein denaturation with urea is due to favorable interaction with the peptide bond rather than to disrupting hydrophobic bonds. In contrast to several recent experiments, however, the exclusion of several protein structure stabilizers (betaine glycine, proline, sorbitol, and trimethylamine oxide) from methyl groups is at least as strong as their estimated exclusion from the peptide backbone. As with the exclusion of nonpolar solutes from charged DNA, the distance dependence of exclusion indicates these polar osmolytes are interacting with nonpolar surfaces through a water structuring force. ? DNA packaging in bacterial viruses: The genomes of many bacteriophages are packaged in protein capsids under a hydrostatic pressure that balances both interhelical forces and bending free energies resulting from coiling DNA in such a tight space. We have collaborated with Knobler and Gelbart (UCLA) to determine the relative contributions of DNA-DNA forces and bending. Partial ejection of phage DNA can be achieved through an osmotic pressure gradient between the external solution and the viral capsid interior. At equilibrium, the energy gained in releasing DNA from the capsid is balanced by the osmotic work done in replacing the DNA with water from the external solution. The distance between DNA helices remaining in the phage head can be inferred from the amount of DNA left and compared with the spacing between helices in assemblies formed without a bending stress at the same osmotic pressure. Quite surprisingly and contrary to expectations, bending does not seem to contribute significantly to the pressure of capsid confined DNA. It is possible, however, that the constraint of fluctuations due to the capsid wall decreases interhelical forces. Further experiments are planned to observe directly by x-ray scattering the spacing between helices of DNA remaining in the phage head as dependent on external osmotic pressure. ? Single Molecule Measurements: We are currently undertaking single molecule measurements of DNA looping kinetics both with and without configurational constraints. Since transcription of many genes is controlled by the formation of DNA loops stabilized by protein-protein contacts, these experiments are relevant for understanding regulation of gene expression. In contrast to the wealth of information on equilibrium thermodynamics of loop formation surprisingly little is known about the kinetics of the process. We are developing two other novel optical tweezer techniques. One is a single particle electrophoresis assay that we are using to characterize polyethylene glycol stabilized charged liposomes used for drug delivery. The other is a new assay to measure conformational rotations of single molecules. Preliminary experiments are being conducted with a model DNA stem-loop system. This is expected to be useful for other systems including an HIV protein that is expected to undergo a molecular rotation upon fusion. ? Structural probes of protein associated water: The exclusion of small solutes from proteins offers new opportunities for probing protein-water structure using small angle neutron scattering (SANS). The protein, bulk solute/water solution, and protein associated water all have different scattering contrasts and contribute separately to the observed scattering intensity. Careful measurement of the zero angle intensity and the radius of gyration as dependent on solute concentration can allow an estimate of the number of protein associated waters and their general location in the protein (surface vs. internal). We have applied this approach to lysozyme that has mainly surface waters and to guanylate kinase that has a large internal water cavity that closes with ligand binding in addition to surface waters.? EcoRI sliding rates: Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then linearly diffusing along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. There have been few measurements of the sliding rate or its dependence on salt concentration or osmotic pressure. Our extensive measurements of EcoRI dissociation rates and specific-nonspecific relative binding constants enable us to determine EcoRI sliding rates from the ratio of dissociation rates of EcoRI from DNA fragments containing one and two specific binding sites. By varying the distance between the two binding sites we are able to confirm a simple linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure indicating the protein moves smoothly along the DNA certainly not hopping on and off as has been suggested. EcoRI is able to diffuse an average of 100 base pairs along DNA in about 10 milliseconds. This rate is about 100-fold slower than the diffusion of the free protein in water indicating that the water at the protein-DNA interface is structured and diffusion rate limiting.
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