Millisecond proteins

The first millisecond protein HX measurements were made by automated quench-flow pulse labeling and were aimed at characterizing early protein-folding intermediates [15, 16]. In these experiments, unlabeled protein was mixed with D O and incubated for a short period (ms), followed by a rapid pH drop and flash freeze to quench the reaction. Labeled samples were then analyzed by NMR (this was 1988, the same year that John Fenn showed the first electrospray protein mass spectra at the American Society for Mass Spectrometry meeting). Quench-flow HX for protein folding was translated to MS a few years later by Miranker and coworkers [17]. 

ESI, LC Monitoring sub-millisecond protein folding, rapid mixing, oxidative labeling Vahidi et a/. [327]... 

The previous application — in accord with most MD studies — illustrates the urgent need to further push the limits of MD simulations set by todays computer technology in order to bridge time scale gaps between theory and either experiments or biochemical processes. The latter often involve conformational motions of proteins, which typically occur at the microsecond to millisecond range. Prominent examples for functionally relevant conformatiotial motions... 

The NMR study by Wiithrich and coworkers has shown that there is a cavity between the protein and the DNA in the major groove of the Antennapedia complex. There are several water molecules in this cavity with a residence time with respect to exchange with bulk water in the millisecond to nanosecond range. These observations indicate that at least some of the specific protein-DNA interactions are short-lived and mediated by water molecules. In particular, the interactions between DNA and the highly conserved Gin 50 and the invariant Asn 51 are best rationalized as a fluctuating network of weak-bonding interactions involving interfacial hydration water molecules. 

Vilardaga, J. P., Bunemann, M., Krasel, C., Castro, M. and Lohse, M. J. (2003). Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat. Biotechnol. 21, 807-12. 

It is, however, important to note what might be accomplished if the growth rate calculated above can be continued for another 10-20 years. If this happens, simulations on proteins with 100-200 residues can be expected to reach into the millisecond domain, and simulations covering a full second would be attainable within about twenty years. Recent demonstrations of the accuracy of modern molecular force fields [62,63] hold the promise that unbiased molecular dynamics simulations could follow the folding process all the way from the completely unfolded state to the native state, a truly exciting prospect. 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

Essentially nothing is known about tyrosine phosphorescence at ambient temperatures. In frozen solution, tyrosine residues have a phosphorescence decay of seconds. We would expect, however, a decay of milliseconds or shorter at ambient temperature. Observation of tyrosine phosphorescence from proteins in liquid solution will undoubtedly require efficient removal of oxygen. Nevertheless, it could be fruitful to explore ambient temperature measurements, since the phosphorescence decay could extend the range of observation of excited-state dynamics into the microsecond, or even millisecond, time range. 

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