Calculation of Symmetric Oligomer Structures from NMR Data

The size range of proteins amenable to NMR spectroscopy has extended to the point where protein oligomer structures are now being routinely determined. Many are symmetric; we found 36 symmetric oligomers solved by NMR in the present protein structure database: 32 dimers, 2 tetramers, 1 pentamer, and 1 hexamer. Hence, we anticipate that an increasing number of symmetric oligomer structures will be studied in the future. Since symmetry-related nuclei have degenerate chemical shift, the resonance assignment problem for symmetric oligomers is simplified compared with asymmetric molecules of similar size. However, the NOESY assignment and structure calculation are much more difficult, mainly due to difficulty in distinguishing among intra-, inter-, and comonomer (mixed) NOE signals. For dimers, this difficulty can be overcome using asymmetric labeling, but ambiguity remains for higher-order oligomers. In this chapter we focus on a calculation method, called the symmetry-ADR method, that we have developed for overcoming these difficulties. The main features of the method are the use of special restraints to specify the oligomeric symmetry, the use of ambiguous distance restraints (ADRs) to represent the ambiguous NOEs, and the use of novel annealing protocols for the structure calculation. We discuss in detail several structure calculations we have made with this method. We also briefly review the structure calculation methods used in all of the symmetric oligomers solved by NMR to date; the majority have been solved by using aspects of the symmetry-ADR method. We conclude that the symmetry-ADR method has proven to be useful and capable of producing accurate structures. However, our experience cautions us that the calculation of symmetric oligomers by NMR remains challenging, particularly for higher-order oligomers.