Location of platinum binding sites on bacteriorhodopsin by electron diffraction

Abstract

A platinum-containing derivative of bacteriorhodopsin has been prepared by treating purple membranes with glycyl-L-methionatoplatinum. Low-dose electron diffraction was used to identify Pt binding sites in the 5.6 A resolution reconstruction of the bacteriorhodopsin unit cell in projection. This is a necessary first step in the use of the Pt derivative for identifying the parts of the amino acid sequence corresponding to the a helices in the bacteriorhodopsin structure and for obtaining phases for reflections out to 3.5 A resolution by the method of heavy-atom isomorphous replacement. The largest peak in a Fourier difference map between platinum-labeled and native purple membrane is larger than the spurious features expected to arise from errors in measurements of diffraction intensities.A number ofproteins that have biologically interesting'functions have been found to be capable offorming two-dimensional crystalline arrays, but not, as ofyet, well-ordered three-dimensional crystals (1-6). Electron diffraction and electron imaging have been used in the structural analyses of these proteins because of the difficulty of obtaining single-crystal x-ray diffraction patterns from two-dimensional small crystals. We discuss here the use of single heavy-atom labels for obtaining detailed structural information from two-dimensional arrays, using as a demonstration our studies ofPt binding to bacteriorhodopsin (BR), the light-driven proton pump from the purple membrane fraction of Halobacterium halobium.The tendency ofBR to form two-dimensional crystals allowed Henderson and Unwin to use low-dose electron imaging and diffraction to determine the three-dimensional structure of the molecule to 7 A resolution (1, 7). Two main factors have blocked the extension ofthis structural study to higher resolution. First, it has been difficult to obtain significant intensity in high-resolution diffraction orders. Because purple membranes are ordered to at least 3.5 A resolution (8), intensity is limited by the low levels of electron irradiation that can be tolerated without destruction of the specimen and by the small size of the crystalline membrane patches. Second, currently available electron imaging systems are incapable ofachieving 3.5 A resolution over large specimen areas without distortion (9).As the amino acid sequence of BR is known (10, 11), one way to get a detailed understanding of BR chain folding is to attach heavy atoms to known amino acids in the sequence and use electron diffraction to locate the heavy atoms in the low-resolution structure. The high a helix content of the protein has already led to some model building efforts (12), a major remaining uncertainty being the question of which part of the sequence is identified with each a helical segment in the structural map.

The opsin family of proteins

“…Establishment ofhelix connectivity has been attempted, partly by elimination of unlikely alternatives on the basis of required lengths for the connecting loops , partly by obtaining higher resolution structures through image enhancement or different crystal forms (Agard & 1A Henderson, 1983) and partly by determining difference diffraction patterns from protein modified at various specified positions (Dumont et al, 1981;Wallace & Henderson, 1982b;Katre et al, 1984). The most widely accepted arrangement is used in the later model (see Fig.…”

Location of an extrinsic label in the primary and tertiary structure of bacteriorhodopsin

“…Therefore, the multiple isomorphous replacement method (MIR) (23, 24) commonly used for protein crystallography may provide a means for phase determination to the highest resolution. We tested an MIR approach using the platinum derivative of Dumont et al (25) at 5.5 A resolution, and our Hg0.6PG site. The MIR phases out to 7 A were on average 630 from the other phase sets; from 7 to 5.5 A, the MIR phases are as different from either set as the other two sets are from each other (-800).…”

X-ray diffraction in temporally and spatially resolved biomolecular science

“…24 It may not be possible to make de novo, high resolution protein structure determinations unless accurate isomorphous derivative intensity differences can be measured. 25 However molecular replacement is now the predominant technique in protein crystallography not least due to the fact that the protein data bank contains more and more of the protein folds seen in nature (the PDB now contains over 100 000 entries). Thus alpha-crustacyanin might yet be solvable with electron (microcrystal) crystallography and using the beta-crustacyanin X-ray crystal structure determined with a xenon heavy atom derivative and soer X-rays from the Daresbury SRS.…”