TOMA DE CONCIENCIA

Hydrogen exchange is a labeling method. Most labeling techniques involve covalent modification of protein structure, often introducing new functional groups and can have undesirable side effects; hydrogen exchange involves the simplest modification possible, the addition of a single neutron. The technique is minimally invasive. Furthermore, exchange experiments may be done in both directions, H-to-D and D-to-H to verify that the label has no meaningful influence on the system. Additionally, because each amino acid is independently labeled, HX measurements principally provide structural information for each residue in the polypeptide. Other labeling techniques are typically restricted in resolution because labeling generally involves only a subset of residues in the protein and many different labeling strategies must be combined to provide a global picture. These features set HX apart from all other labeling techniques.

2.2.1 Native State Hydrogen Exchange (NHX-type)

“A protein cannot be said to have ‘a’ secondary structure but exists mainly as a group of structures not too different from one another in free energy… the molecule must be conceived as trying out every possible structure each in accordance with its Boltzmann factor.” This statement was written in 1959 by K.U. Linderstrom-Lang and John Schellman (136) working together on protein hydrogen exchange and long before the exact types of motion that determine HX behavior were known.

19 The NHX namesake arises because of collecting these experiments under conditions where all molecules have structurally equilibrated and often where the native state is favored. In such conditions, the ensemble average conformation is native; however, all molecules are continuously exploring higher energy, exchange competent forms and the frequency of these deviations are reflective, under EX2 conditions, of the free energies of local structure. Structures with lower stability exchange faster than those with higher stability.

NHX experiments have been successfully utilized to study unfolding reactions of proteins, notably, the method was used to structurally characterize independently unfolding subunits of structure in a protein that optically unfolds in a two state manner (9). NHX experiments are also used to characterize structural changes that result from ligand binding. In one example for a lipid binding protein (146), regions where labeling rates change in the presence of lipid provide information on the induction of structure by binding. In Chapter 3, NHX experiments on MBP analyzed by NMR are discussed briefly.

2.2.2 Pulse-Labeling Hydrogen Exchange (KHX-type)

Kinetic pulse-labeling experiments (KHX) provide time-resolved insight into temporally fleeting events that are not accessible via the NHX method. In contrast to NHX where the experiment is conducted under equilibrium conditions, KHX experiments are generally synchronized-start experiments and involve interrogating a system as it relaxes to equilibrium. KHX has traditionally been used for studying protein folding reactions; but has also been used for more exotic purposes such as studying conformational changes occurring during the catalytic cycle of chymotrypsin (147).

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Figure 2.1: The kinetic pulse labeling HX experiment. The protein is fully unlabeled in the unfolded state before diluting into permissive refolding conditions. At a variable time during folding, the HX pulse is applied. This is analogous to spray-paint that will not stick to hydrogen bonded residues, such as those in the helical conformation above. Following the pulse, for MS analysis, the sample is digested by proteases and the fragments separated, this is described in Chapter 4, page 51. For NMR analysis, the sample is allowed to continue folding until the native structure is acquired. Those regions with no paint were folded at the time of the pulse.

KHX experiments, in the context of protein folding investigations, are typically performed with a rapid mixing device to facilitate fine control over labeling times. Starting from a chemically unfolded state, folding is initiated by dilution of denaturant to permissible folding conditions where exchange is neutralized. Folding is allowed to proceed for a variable amount of time before rapidly exchanging solvents, usually by rapid dilution, into a brief, high pH condition where exchange is accelerated. This is quite analogous to the application of spray-paint only to those regions of structure that are not H-bonded, as is illustrated in Figure 2.1. This process results in a labeling pattern which reports on the structure that formed during the folding phase, before application of the labeling pulse.

Under favorable conditions where both structural opening and closing reactions are negligible over the period of the pulse, KHX is a binary experiment and easily interpreted. Amide protons that develop H-bonded structure during the folding phase, such as in an intermediate, are protected from exchange whereas sites that remain unfolded will label to completion during

21 the pulse. Structural information may then be directly inferred from the time dependence of H- bonding. Regions of the sequence involved in intermediate structures will display protection from labeling on a similar time scale.

If structural opening reactions occur on the time scale of the pulse, residues that would have been completely protected during the pulse may lose some label and introduce a second order effect on the measurement. In these situations, changing the pulse length, pH, or temperature can each provide information on the equilibrium constant for H-bonding at each protected site and allow one to draw inferences regarding structural heterogeneity. In Chapter 5, I characterize an obligatory intermediate in the folding pathway of MBP using the binary experiment and discover heterogeneous structural features of the MBP burst-collapse event using variable pulse lengths at a fixed folding time.