NIH's Ad Bax Takes NMR Spectroscopy to New Demensions

That's not particularly complex for a protein, is it?

By 1980 standards, it was forbiddingly complex. It had been studied by a number of groups and pretty much shown to be impossible. That's why we picked it.

So demonstrating that you could do it must have made quite a splash.

That was a big hit as far as citations. We first showed we could do the resonance assignment process, figuring out which nucleus resonates at which frequency by a new approach we developed known as triple resonance assignment. That allowed us then to identify all the resonance frequencies in this calmodulin protein. That assignment paper came out in 1990. The main question left, the structure of the protein complexed to a fragment of its target, was published in 1992.

You had a particularly hot streak of highly cited papers between 1990 and 1992. How much could you do in 1992 that you couldn't when you first came to NIH?

A hell of a lot more. Nevertheless, in 1992, it was still a formidable challenge to do a larger protein. We were the first to solve a 20-kilodalton protein by NMR. That was in 1992.
Now we can determine the structures of smaller proteins, up to about 20 kilodaltons, relatively routinely. There must be at least several hundred that have been solved in this manner. It takes about half a year to get a reasonable or medium-quality structure for it. Then, with some additional work, with another half year or so, one can get quite a good structure. Additionally, we can study the mobility within the protein. Proteins are not bricks; they have a lot of flexibility built into them, which is essential for them to function. NMR allows us to look at this mobility in a great amount of detail. That is where I anticipate that considerable technological developments will allow us to yield more detail than what we've been able to get so far.

Ad Bax's Highest-Impact Papers
Published Since 1990
(Ranked by average citations per year)

Rank	Paper	Total Citations	Average cites per year
1	M. Ikura, et al., "Solution structure of a calmodulin-target peptide complex by multidimensional NMR," Science, 256(5057):632-8, 1992.	401	73
2	L.E. Kay, et al., "Three-dimensional triple resonance NMR-spectroscopy of isotopically enriched proteins," J. Magn. Res., 89(3):496-514, 1990.	284	44
3	M. Ikura, L.E. Kay, A. Bax, "A novel approach for sequential assignment of H-1, C-13, and N-15 spectra of larger proteins: Heteronuclear triple resonance three-dimensional NMR spectroscopy. Application to calmodulin," Biochem., 29(19):4659-6, 1990.	318	42
4	A. Bax, et al., "Comparison of different modes of two-dimensionalreverse-correlation NMR for the study of proteins," J. Magn. Res., 86(2):304-18, 1990.	316	42
5	A. Grzesiek, A. Bax, "The importance of not saturating H₂O in protein NMR: Application to sensitivity enhancement and NOE measurements," J. Amer. Chem. Soc., 115(26):2593-4, 1993.	144	41

SOURCE: ISI's Science Indicators Database, 1990-1997

What are the largest proteins that can be done now?

The maximum size that can be addressed today with reasonable quality is maybe 30 kilodaltons. That really is still the limit for single-chain proteins. We can do dimeric proteins a little bit larger. Beyond that we can still get resonance assignments, but the structural quality becomes more like an approximate cartoon rather than a well-defined, high-resolution structure. It all depends how much time, effort, and money you're willing to spend.

Where does the field go from here?

We've been working on interesting new methods that are going to put us quite a few steps ahead again. We just published a paper, for instance, where we showed that we could get a very different type of information by using the fact that proteins orient themselves very weakly in strong magnetic fields. It's known as dipolar coupling.

This method tells us the orientation of every pair of atoms for which we can see a dipole coupling: what the orientation of this pair of atoms is relative to a given axis system. This is useful because we can determine this orientation for all pairs relative to a single axis system, which means that even pairs of atoms on opposite ends of the protein are still aligned relative to the same axis system. So we get really long-range information that's not accessible in conventional, old-style NMR experiments.
Following up on that, we had a recent paper in Science where we increase this ability to align proteins by demonstrating that you can dissolve proteins in a liquid crystalline phase and get them to orient that way (see N. Tjandra and A. Bax, Science, 278[5340]:1111-4, 7 November 1997). This makes the method very general, and will allow us to improve the quality of NMR structures tremendously. It really looks extremely promising. Even though we will need to measure more parameters, it appears we will be able to complete the protein structure determination far faster than we could before.

Do you feel like making a prediction about what will be possible in five years?

Well, it's always hard to predict. What I expect, at least, is for researchers to generate very high-quality structures by NMR that are comparable to very good crystal structures in terms of accuracy. But, in addition, they will really have the characterization of the protein in free solution. It will become more meaningful to compare the difference between solution and crystalline state, allowing us to address more subtle questions than, say, what is the fold of a given protein. We may be able to study inter-molecular interactions at an exquisite level of detail. For example, protein-protein interactions at a level of detail that has not been even close to being achieved satisfactorily with the present methodology.

Is there one particular problem you dream about solving someday?

My big goal is to study intact integral membrane proteins by NMR. That can't be done now. These proteins are, of course, very important in virtually all diseases, ranging from cancer to arthritis to diabetes to you name it. That's because the signaling for what a cell is supposed to do always comes from the outside and goes in to the cell. If anything gets messed up at the membrane boundary surrounding the cell, it has a devastating effect on the organism. But it's almost impossible to get structural information on these proteins. They are very difficult to crystallize. If we could do it, it would yield an enormous amount of biomedically relevant information.

What do you think it will take?

It's hard to tell. New methods-which I can't really describe in any more detail. All I can say is that it's a long shot, but I don't think the situation is hopeless.