Assembly of a Polytopic Membrane Protein Structure from the Solution Structures of Overlapping Peptide Fragments of Bacteriorhodopsin

Abstract

Three-dimensional structures of only a handful of membrane proteins have been solved, in contrast to the thousands of structures of water-soluble proteins. Difficulties in crystallization have inhibited the determination of the three-dimensional structure of membrane proteins by x-ray crystallography and have spotlighted the critical need for alternative approaches to membrane protein structure. A new approach to the three-dimensional structure of membrane proteins has been developed and tested on the integral membrane protein, bacteriorhodopsin, the crystal structure of which had previously been determined. An overlapping series of 13 peptides, spanning the entire sequence of bacteriorhodopsin, was synthesized, and the structures of these peptides were determined by NMR in dimethylsulfoxide solution. These structures were assembled into a three-dimensional construct by superimposing the overlapping sequences at the ends of each peptide. Onto this construct were written all the distance and angle constraints obtained from the individual solution structures along with a limited number of experimental inter-helical distance constraints, and the construct was subjected to simulated annealing. A three-dimensional structure, determined exclusively by the experimental constraints, emerged that was similar to the crystal structure of this protein. This result suggests an alternative approach to the acquisition of structural information for membrane proteins consisting of helical bundles.

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

“…Therefore, it is reasonable to presume that they would also be favorable within the setting of the NHE1 protein. Indeed, a number of studies have demonstrated strong correspondence between structures of peptides or protein segments obtained in membrane mimetic solvents to structures obtained by solution state NMR in micelles or to entire membrane proteins determined by x-ray crystallography (11)(12)(13). Most interestingly, we find that CD 3 OH:CDCl 3 :H 2 O provides a well defined peptide structure, whereas the peptide in Me 2 SO appears much less structured.…”

“…This provides insight into structured regions, in our case, without fixing the segment as a whole into a single conformation. The use of solution conditions having a low dielectric constant to mimic a membrane environment has become quite common for structural studies of transmembrane proteins or peptides and has provided structures of isolated protein segments consistent with their structures in the full protein (11)(12)(13). Our study demonstrates that whereas TM IV is structured, only a 4 -6-residue stretch of the segment is helical.…”

Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2*

“…Earlier studies have demonstrated that the amino acid sequences of TM segments of membrane proteins contain most of the required structural information needed to form their native structures (55)(56)(57)(58). This approach has been used successfully previously for mammalian NHE1 (15,16) and for the membrane domain of CorA (59).…”

Structural and Functional Characterization of Transmembrane Segment VII of the Na+/H+ Exchanger Isoform 1

“…It must be acknowledged that an isolated membrane segment could have a different structure than that found in an intact membrane protein. However, isolated TM segments from membrane proteins having multiple TM segments, including the cystic fibrosis transmembrane conductance regulator (17,18), bacteriorhodopsin (19,20), rhodopsin (21), and the fungal G-protein-coupled receptor Ste2p (22), have been shown to be both functional and to have structures in good agreement with the segments in the context of the entire protein, where available. An important caveat is that solution conditions may need extensive screening in order to achieve stabilization of the peptide in its physiological structure, for example, the variation in bacteriorhodopsin results of Ref.…”