The protein in bulk solution is less than 5%, and within the uncertainties of the d2D method. No increases in a-helical content are AbMole Benzyl alcohol observed that would be indicative of the rapidly reversible population of oligomeric or membrane-associated states, although our observations do not preclude the existence of an NMR-invisible membrane-bound sub-population of aSyn in slow exchange with the disordered state. Although the chemical shift changes are small, and no change in the secondary structure content of aSyn is detectable within the cell, some differences are nevertheless apparent in the spectra, notably marked intensity changes in the HNCO spectrum of intracellular aSyn relative to the bulk solution state. Decreased intensities are observed over much of the sequence, particularly in the region of the N and C-termini, and indeed as a result of this broadening no Ca and Cb resonances could be detected between residues 1 and 26. Such peak broadening could arise from intermediate chemical exchange, indicating conformational fluctuations on a millisecond timescale, which has been observed previously in NMR studies of binding interactions involving aSyn. In particular, decreased intensities within the N-terminal domain suggests that interactions may be occurring with the cell membrane, as similar intensity changes have previously been observed for the isolated protein in the presence of model membrane systems. Within the cell however, line broadenings can also be due to transferred relaxation, as a result of weak and transient interactions with other large and slowly tumbling macromolecules within the crowded cellular environment; indeed the highly charged nature of the N and C-terminal regions of aSyn may result in a particular propensity for non-specific electrostatic interactions. In summary, we have demonstrated that a multidimensional reference deconvolution strategy can substantially decrease the inhomogeneous line broadening associated with cellular samples. Using this approach, backbone chemical shifts have been measured for samples of aSyn expressed within bacterial cells, and used to evaluate secondary structure formation in this environment. Although selective reductions in peak intensity are observed, indicative of interactions with other components of the cell, only small chemical shift differences are observed compared with monomeric aSyn in bulk solution, indicating that in the crowded cytosolic environment the protein exhibits a disordered conformation whose secondary structure closely resembles that observed in studies of aSyn in dilute aqueous solution. More generally, given the increasingly recognized importance of intrinsically disordered proteins or domains in many cellular processes, we believe that the approach we have described here will become an important method to investigate the structure and behavior of such molecules directly within the cellular environment.