Professional interests || Personal interests|| Contact

Some Millhauser lab references.

Some references from other labs.

Nitroxides are stable organic free radicals which possess an unpaired electron. The magnetic resonance signal of this electron can be detected and characterized by ESR (Electron Spin Resonance spectroscopy). This signal can provide information about the motion, distance, and orientation of unpaired electrons in the sample with respect to each other and to the external magnetic field. For molecules free to move in solution, ESR works on a much faster time-scale than NMR (Nuclear Magnetic Resonance spectroscopy), and so can reveal details of much faster molecular motions--nanoseconds as opposed to microseconds for NMR. The gyromagnetic ratio of the electron is orders of magnitude larger than of nuclei commonly used in NMR, and so the technique is more sensitive, though it does require spin labeling.

For spin-spin distance measurements analogous to the nOe experiments, this translates into the ability to measure distances longer than those practically accessible by direct NMR measurement, and shorter than those for which fluorescence energy transfer is useful.

                technique       useful distance range (angstroms)
                NMR                         < 6.5
                ESR                      5 - 12
                FET                     10 - 80


Finally, it's relatively much simpler to look at spin-labeled samples in restricted environments (solid state, aggregates, ordered environments, etc) for ESR than for NMR. For example, one can use the same spectrometer to look at the same sample as spin label in the sample moves from solution into an aggregate. Solution NMR and solid-state NMR typically require different spectrometer hardware and very different sample preparations, in contrast. Wayne Hubbell at UCLA has used spin labels, for example, to probe structural details of membrane-bound proteins like bacteriorhodopsin and lac permease, and Glenn has recently published a graduate student's work which showed the uptake of a soluble, spin labeled monomer by fibrillar structures similar to those seen in amyloid diseases.

The dominant method in the literature today for site-specifically labelling protein sequences is the reaction between methanethiosulfonate spin label and cysteine, to give the spin-labelled cysteine side chain, CYS-SL:

        MeS(O)2SSR + R'SH  --->  R'SSR + MeS(O)2SH

where R is the nitroxide group and R'SH is a protein with a cysteine sulfhydryl, and R'SSR is the spin-labeled protein. The cysteines for labelling are placed in the desired sequence position either through solid-phase techniques or through standard recombinant DNA techniques.

There are a number of drawbacks to this approach, however, which motivates the development of other labeling techniques. Obviously, in proteins in which disulfides are dominant functional or structural elements one wouldn't want to risk gross perturbations by messing around with the cysteines. Another disadvantage is the chemical lability of the disulfide in the CYS-SL sidechain--there's a fairly restrictive temperature range over which we've been able to use CYS-SL, especially in double-label experiments. Yet another consideration is that we'd like to have broader choice in the nature of the tether which ties the spectroscopic reporter group to the peptide backbone. For example, CYS-SL is a fairly long, flexible tether, while TOAC offers a much shorter tether, but at the expense of non-neglible steric effects at the backbone.


For some line drawings of some interesting spin-label amino acids, check here.


last modified by dja on 20090120