MAQUINA/EQUIP O REGISTRO FOTOGRÁFICO

HX MS provides an opportunity to independently monitor the partition of molecules between definable non-degenerate conformational states. State sensitivity is essential for discerning between multiple pathway models such as IUP and sequential pathway models such as PPOE. There are an exceedingly small number of ways to achieve state-sensitive measurements. To our knowledge, HX MS is the only direct approach. By using an example from the MBP pulse labeling HX MS dataset (presented in Chapter 5), this exceptionally rare capability is demonstrated here.

Figure 2.2: State sensitivity in HX MS experiments. Spectra are shown at different refolding times for three MBP peptides. Three states appear due to the presence of an obligatory intermediate (7 second li

peptide 21-43 and not peptide 44

to two states with this approach. If states are degenerate with respect to the number of H indistinguishable; states are also hard to resolve if they are not sufficiently separated by mass.

In KHX experiments, we interpret protection from exchange as representative of H bonded structure that formed prior to application of the labeling pulse (or spra

2.1). Because mass measurements are not ensemble HDpop program, described on page

state sensitivity; this is illustrated in

populations whose sum is traced in red. The area contained within each blue dashed line represents the relative population fraction of molecules in the pa

the average number of deuterium retained.

The KHX experiment, a focus of this dissertation, tends to have a binary nature with respect to stable H-bonded structure formed before the labeling pulse. For example, if an

: State sensitivity in HX MS experiments. Spectra are shown at different refolding times for three MBP peptides. Three states appear due to the presence of an obligatory intermediate (7 second li

43 and not peptide 44-61. A peptide covering both is also shown to demonstrate that we are not limited to two states with this approach. If states are degenerate with respect to the number of H-bonds, they will appear

tinguishable; states are also hard to resolve if they are not sufficiently separated by mass.

In KHX experiments, we interpret protection from exchange as representative of H bonded structure that formed prior to application of the labeling pulse (or spra

). Because mass measurements are not ensemble-averaged in the classical sense, using program, described on page 73, for analysis of mass distributions provides unbiased state sensitivity; this is illustrated in Figure 2.2 where the blue dashed lines demarcate individual populations whose sum is traced in red. The area contained within each blue dashed line represents the relative population fraction of molecules in the particular HX state, defined by the average number of deuterium retained.

The KHX experiment, a focus of this dissertation, tends to have a binary nature with bonded structure formed before the labeling pulse. For example, if an 22

: State sensitivity in HX MS experiments. Spectra are shown at different refolding times for three MBP peptides. Three states appear due to the presence of an obligatory intermediate (7 second lifetime) that contains 61. A peptide covering both is also shown to demonstrate that we are not limited bonds, they will appear

In KHX experiments, we interpret protection from exchange as representative of H- bonded structure that formed prior to application of the labeling pulse (or spray-paint in Figure

averaged in the classical sense, using the for analysis of mass distributions provides unbiased where the blue dashed lines demarcate individual populations whose sum is traced in red. The area contained within each blue dashed line rticular HX state, defined by

The KHX experiment, a focus of this dissertation, tends to have a binary nature with bonded structure formed before the labeling pulse. For example, if an

23 initially deuterated 20 residue peptide has 10 protecting H-bonds due to structure that formed in 50% of the molecules prior to pulsing the sample with H2O, we would expect to see two

populations – one with the mass of the unfolded control and the other shifted by roughly 10 Da after back exchange correction. Each population would have an equivalent area reflecting the 50/50 distribution of molecules. Figure 2.2 shows MBP peptide 21-43 who behaves in this manner. This peptide stably forms a tentative helix that protects 12 deuterons with a folding lifetime of 7 seconds; thus, we observe a heavy population who transitions from the unfolded, low mass, distribution to the +12 mass distribution with an appropriate time signature. Similarly, peptide 44-61 also gives a binary result although the transition occurs on a different time scale. The power of HX MS state sensitivity is most clearly demonstrated in peptide 21-61 which clearly shows three populations who transition between one-another on timescales reflective of the two different behaviors in the smaller peptides in Figure 2.2.

Generally, each population in a KHX experiment has a constant mass with respect to folding time; but, if observed, the time dependence of mass changes for a given population may provide useful information. For example, the light population centroid in peptide 44-61 appears to shift by approximately +3 Da between the 0 s and 30 s time points after correcting for back exchange. Upon quantitation of the time dependence, we observe this mass shift to coincide with the bulk population transition shown in peptide 21-43. This can occur when two populations are not resolved from one another – in this case, the centroid of the cumulant6 population will move, just as observed in peptide 44-61. We interpret such observations to represent, in most cases, two populations who are unresolved. In this particular example, the mass shift observed in 44-61 represents a β-strand with three H-bonds whose concerted formation with the helix described above and proximity of the residues in the native structure implies both structures form together with a characteristic lifetime of seven seconds. This is explored in detail in Chapter 5.

A binary KHX result, such as the one just described, requires that the opening rate lifetime of protecting structure exceed the duration of the pulse by at least a factor of three;

6

Cumulant – I have defined this to describe two populations who are unresolved from one another. As the fraction of molecules in one unresolved population transition to the other unresolved population, the centroid mass of the cumulant envelope will shift from being dominated by the first to being dominated by the second unresolved mass centroid.

24 otherwise, preformed structure will open in a significant fraction of the molecules during the pulse and subsequently exchange. In this case, there will appear to be fewer molecules in the protected population than actually existed when the labeling pulse was applied. Modulating the pulse duration allows us to test for such behavior during the MBP collapse (p. 91).