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When one looks at a protein structure on a computer screen, one is looking at a static representation of the conformation. This however, is not a true picture of a protein. A protein is more like a piece of jelly floating in the water: it can wobble and distort on microsecond and faster time scales. The flexibility of a protein is dependent on the nature of the internal structure and extrinsic factors such as temperature and solution composition. Because of the small size of proteins, it is difficult to view these motions directly. However, the strongest evidence for protein flexibility has come from the use of NMR to monitor hydrogen exchange.

Rationale for H-exchange

The best evidence for protein movement is that groups buried inside a protein can chemically interact with the solvent. Either the buried group must spend some time at the surface, thereby interacting with the solvent, or the protein must "breathe" in such a way that water can sometimes work its way into the protein. In either case, this means that the rigid, static structure of the protein must give way to change in order to interact with the solvent. Using isotopic forms of water such as D2O - deuterated water, one can look for the appearance of deuterium at internal positions on a protein as evidence of solvent - protein hydrogen exchange.

Methodology

Amide hydrogens (particularly those hydrogens that belong to nitrogens on the polypeptide backbone) are used in exchange measurements, because these hydrogens exchange on a convenient time scale. Furthermore, because they are buried inside the protein, exchange is even slower and easier to measure. Both acid and base catalysis of amide hydrogens are believed to occur in proteins. Catalysis by acid is believed to occur by transient protonation of the backbone carbonyl (C=O) oxygen, followed by a transient loss of the adjacent N-H hydrogen and its replacement from the solvent. In base mediated catalysis, a hydroxide from the solvent can remove a N-H hydrogen directly, subsequently replaced by one from the solvent. The minimum rate of exchange occurs at around pH = 3.

The exchange of individual hydrogen atoms can be followed using 1H-NMR which, because of its high resolution, allows the assignment of resonances to individual protons. This technique has found that protons in the interior of the protein exchange less readily than those on the surface. In secondary structure motifs such as beta-sheets, exchange occurs more readily at the ends rather than the interior.

Protein Breathing

Local unfolding of a protein, or breathing, is often invoked to explain the exchange of interior hydrogens. The classical exchange mechanism would be as follows:
	          k1
	folded  <--->  open -----> hydrogen exchanged
                  k-1         kex
The hypothetical open form is unstable and transient. Therefore, k1 is much smaller than k-1, making the equilibrium far smaller than one. The rates of exchange are generally characterized by the important step. The EX1 mechanism is where the opening rate is the determining factor. If kex >> k-1, then k1 should equal the experimentally observed exchange rate. In an EX2 mechanism, kex << k-1, thereby making the exchange rate sensitive to the equilibrium constant between folding and opening. If an EX2 mechanism is occuring, then the exchange rate should be sensitive to processes that affect the folding equilibrium such as pH. Most proteins exhibit EX2 exchange. EX1 generally occurs only at high pH in proteins, where the proton activity makes hydrogen exchange very fast.

Solvent Permeation

The alternative hypothesis is that water is able to permeate into the protein, exchanging hydrogens. It is believed that quenchers of fluorescence such as acrylamide can diffuse into proteins and disrupt fluorescence of buried tryptophans. The rate of exchange would then depend in a very complex manner on the general flexibility and its ability to create channels in the protein. Observations that support the permeation model are: (1) exchange in a protein crystal are often similar to those observed in free protein: if a crystal lattice constrains breathing, one would not expect this to be so, (2) the rate of exchange is not increased by low concentrations of urea or other denaturing agents which would be expected to shift the equilibrium towards unfolding, and (3) the rate of exchange increases with pressure, suggesting a need to create channels in the protein to allow water to enter.

Although the available evidence is not sufficient to clearly establish breathing over permeation (and maybe both occur), the general model used in the field is one of protein breathing.

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