V. Adrian Parsegian, PhD, Chief

The Laboratory of Physical and Structural Biology (LPSB) brings many science disciplines to bear on biological investigations. As physicists, LPSB investigators consider the next step in structural biology as not simply determining the structure of every identifiable entity from molecule to organelle but rather as learning how these structures work through the physics and chemistry of the intermolecular forces that create them. They learn from the increasing number of protein, nucleic acid, saccharide, and lipid structures to design agents that compete effectively with deviant interactions associated with disease. This year they revealed essential physical features of viral infection, DNA packing, and lipid-deficient disease.

Sergey Bezrukov and his colleagues in the Section on Molecular Transport are monitoring the initial stages of phage lambda infection with the goal of using bacteriophages as agents to control antibiotic-resistant pathogens such as Streptococcus pyogenes, Staphylococcus aureus, and even Bacillus anthracis. They were able to observe the first step in bacteriophage infection, the docking of phage lambda to its trimeric receptor LamB, or maltoporin, at the single-particle level. Reconstituted into a bilayer lipid membrane, the receptor forms stable water-filled pores that conduct ions and sugars. In the absence of substrate (malto-sugars), the three receptor pores are always open, but they are transiently blocked by added permeating maltohexaose. Investigators found that the phage interacts simultaneously with all three monomers of the maltoporin receptor, modifying their transport properties. Remarkably, although ionic conductance drops by at most 30 percent upon phage docking, sugar access from the side of phage addition is completely obstructed. The statistics of maltohexaose binding to the phage-receptor complex on the side opposite phage docking shows that the monomers of maltoporin still bind sugar independently, with the kinetic constants expected for those of the phage-free receptor. This finding suggests that phage docking does not significantly change the structure of the receptor and that phage-binding regions are close to, but do not overlap with, the sugar-binding domains of the receptor monomers. The analysis reveals that ion fluxes through maltoporin pores share a new common pathway in the phage-receptor complex.

Donald Rau, who heads the Section on Macromolecular Recognition and Assembly, conducts single-molecule force measurements of DNA packaging. Highly positively charged proteins such as histones or protamines mediate the compaction of DNA in the cell. DNA packaging by synthetic polycations is central for delivery systems used in gene therapy. The laboratory's measurements suggest that the attractive force between DNA helices mediated by highly charged cations is likely attributable to water structuring rather than to conventional electrostatics. DNA helices precipitated by these multivalent ions are separated by 7-10 Å of water, depending on the particular condensing cation, indicating the presence of both attractive and repulsive force components. To connect attraction and water structuring energetics more conclusively, investigators combined osmotic stress X-ray measurements that probe repulsive forces with single-molecule magnetic tweezer experiments designed to probe attractive forces between DNA helices. The magnetic-tweezer pulling force necessary for preventing the collapse of a single DNA molecule to the equilibrium spacing is a measure of the attractive force. Predictably, the force varies with condensing ion concentration because of differences in the number of ions bound to the condensed and extended states. The single-molecule measurements give the depth of the free energy minimum attributable to net interhelical attraction unmodified by the energies associated with ion rearrangement. Combining single-molecule measurements with osmotic stress measurements of repulsive free energy, the group is able to determine the attractive free energy component itself. For all condensing ions examined, the investigators found a critical ratio of attractive and repulsive free energies that confirms an interpretation based solely on hydration forces.

Adrian Parsegian, who heads the Section on Molecular Biophysics, investigates the molecular forces in molecular disease, in particular the mechanism by which cholesterol affects sterol granule biogenesis in vivo. This past year, the laboratory investigated why removal of a double bond creates a species that frustrates normal vesicular secretion, as occurs in the deadly Smith-Lemli-Opitz syndrome. Absence of one of the last two enzymes in the cholesterol biosynthetic pathway results in an accumulation of precursors. Remarkably, regulated secretion could be restored with exogenous normal cholesterol. The observed reversal with the simple addition of normal cholesterol immediately suggested that physical forces are at work, forces that can differ with small differences in sterols. The laboratory found that modification of the sterol-chemical structure significantly alters membrane physical properties. By measuring changes in the bending rigidity of bilayers containing either cholesterol or one of its metabolic precursors (lanosterol, 7-dehydrocholesterol, or lathosterol), investigators found that cholesterol was the most efficient in enhancing membrane rigidity, a possible clue as to why depletion or replacement with other sterols can affect cellular structures. The stiffness of a granule is likely an important factor in the deformations it must endure to undergo secretion.