Thermoacoustic range verification using a clinical ultrasound array provides perfectly co-registered overlay of the Bragg peak onto an ultrasound image.

The potential of particle therapy due to focused dose deposition in the Bragg peak has not yet been fully realized due to inaccuracies in range verification. The purpose of this work was to correlate the Bragg peak location with target structure, by overlaying the location of the Bragg peak onto a standard ultrasound image. Pulsed delivery of 50 MeV protons was accomplished by a fast chopper installed between the ion source and the cyclotron inflector. The chopper limited the train of bunches so that 2 Gy were delivered in [Formula: see text]. The ion pulse generated thermoacoustic pulses that were detected by a cardiac ultrasound array, which also produced a grayscale ultrasound image. A filtered backprojection algorithm focused the received signal to the Bragg peak location with perfect co-registration to the ultrasound images. Data was collected in a room temperature water bath and gelatin phantom with a cavity designed to mimic the intestine, in which gas pockets can displace the Bragg peak. Phantom experiments performed with the cavity both empty and filled with olive oil confirmed that displacement of the Bragg peak due to anatomical change could be detected. Thermoacoustic range measurements in the waterbath agreed with Monte Carlo simulation within 1.2 mm. In the phantom, thermoacoustic range estimates and first-order range estimates from CT images agreed to within 1.5 mm.

Crystal structure of an engineered Cro monomer bound nonspecifically to DNA: possible implications for nonspecific binding by the wild-type protein.

The structure has been determined at 3.0 A resolution of a complex of engineered monomeric Cro repressor with a seven-base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half-operator that is recognized by each subunit of the wild-type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence-independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48 degrees further out of the major groove, while the turn region of the helix-turn-helix remains in contact with the DNA backbone. All of the direct base-specific interactions seen in the wild-type Cro-operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein DNA interface than for half of the wild-type Cro-operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence-nonspecific interaction of the wild-type Cro dimer with DNA. In the sequence-specific complex, the DNA is bent, the protein dimer undergoes a large hinge-bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.

Crystal structure of lambda-Cro bound to a consensus operator at 3.0 A resolution.

The structure of the Cro protein from bacteriophage lambda in complex with a 19 base-pair DNA duplex that includes the 17 base-pair consensus operator has been determined at 3.0 A resolution. The structure confirms the large changes in the protein and DNA seen previously in a crystallographically distinct low-resolution structure of the complex and, for the first time, reveals the detailed interactions between the side-chains of the protein and the base-pairs of the operator. Relative to the crystal structure of the free protein, the subunits of Cro rotate 53 degrees with respect to each other on binding DNA. At the same time the DNA is bent by 40 degrees through the 19 base-pairs. The intersubunit connection includes a region within the protein core that is structurally reminiscent of the "ball and socket" motif seen in the immunoglobulins and T-cell receptors. The crystal structure of the Cro complex is consistent with virtually all available biochemical and related data. Some of the interactions between Cro and DNA proposed on the basis of model-building are now seen to be correct, but many are different. Tests of the original model by mutagenesis and biochemical analysis corrected some but not all of the errors. Within the limitations of the crystallographic resolution it appears that operator recognition is achieved almost entirely by direct hydrogen-bonding and van der Waals contacts between the protein and the exposed bases within the major groove of the DNA. The discrimination of Cro between the operators OR3 and OR1, which differ in sequence at just three positions, is inferred to result from a combination of small differences, both favorable and unfavorable. A van der Waals contact at one of the positions is of primary importance, while the other two provide smaller, indirect effects. Direct hydrogen bonding is not utilized in this distinction.

How Cro and lambda-repressor distinguish between operators: the structural basis underlying a genetic switch.

Knowledge of the three-dimensional structures of the lambda-Cro and lambda-repressor proteins in complex with DNA has made it possible to evaluate how these proteins discriminate between different operators in phage lambda. As anticipated in previous studies, the helix-turn-helix units of the respective proteins bind in very different alignments. In Cro the recognition helices are 29 A apart and are tilted by 55 degrees with respect to each other, but bind parallel to the major groove of the DNA. In lambda-repressor [Beamer, L. J. & Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196] the helices are 34 A apart and are essentially parallel to each other, but are inclined to the major grooves. The DNA is much more bent when bound by Cro than in the case with lambda-repressor. The first two amino acids of the recognition helices of the two proteins, Gln-27 and Ser-28 in Cro, and Gln-44 and Ser-45 in lambda-repressor, make very similar interactions with the invariant bps 2 and 4. There are also analogous contacts between the thymine of bp 5 and, respectively, the backbone of Ala-29 of Cro and the backbone of Gly-46 of lambda-repressor. Otherwise, however, unrelated parts of the two proteins are used in sequence-specific recognition. It appears that similar contacts to the invariant or almost invariant bps (especially 2 and 4) are used by both Cro and lambda-repressor to differentiate the operator sites as a group from other sites on the DNA. The discrimination of Cro and lambda-repressor between their different operators is more subtle and seems to be achieved primarily through differences in van der Waals contacts at bp 3', together with weaker, less direct effects at bps 5' and 8', all in the nonconsensus half of the operators. The results provide further support for the idea that there is no simple code for DNA-protein recognition.

High-resolution structure of an engineered Cro monomer shows changes in conformation relative to the native dimer.

A rationally designed, genetically engineered, monomeric form of the Cro protein from bacteriophage lambda has been crystallized and its structure determined by isomorphous replacement and refined to a resolution of 1.54 A. The structure confirms the rationale of the design but, at the same time, reveals 1-2 A shifts throughout the monomer structure relative to the previously determined structure of the dimeric wild-type protein. These changes include a 1.6 A main-chain shift in part of the beta-sheet region of the molecule relative to the alpha-helical region and a 1.1 A shift of a buried phenylalanine within the core as well as a correlated 2.2 A shift in a solvent-exposed beta-hairpin. The conformational adjustments appear to reflect an inherent flexibility of the protein that is associated with its DNA-binding function.

Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis.

The crystal structure of an engineered monomer of the lambda Cro repressor shows unexpected expansion of the hydrophobic core of the protein and disorder of the five C-terminal residues [Albright et al. (1996) Biochemistry 35, 735-742]. This structural information has guided the construction of a second generation of monomeric Cro proteins by combinatorial mutagenesis of selected core and C-terminal residues. Clones were identified in a library of randomized cro genes by a genetic screen for protein accumulation in Escherichia coli. Sequencing of candidate genes followed by purification and analysis of their product proteins has identified alternative arrangements of hydrophobic core residues which result in substantial increases in thermal stability. In contrast, residue replacements at the C-terminus have minor effects on stability but may increase protein expression levels.