It appears that no major hydration-induced conformational changes occur at hydrations above about 0.2 h. For example, the constancy of hydrogen exchange rates above about 0.15 h  and the detection of enzymatic activity in a number of enzymes at hydrations of 0.12-0.2 h [39,41-43] would suggest that the protein conformation above hydration levels of about 0.2 h is essentially the same as that in dilute solution. Other experimental evidence supporting this conclusion has been summarized by Rupley et al.  and Luscher-Mattli and Ruegg .
Although the appearance of hysteresis in the sorption isotherms, as well as a variety of spectroscopic evidence, suggests that conformational changes can occur at hydration levels below 0.2 h, the extent of the structural changes that do occur has been a matter of some debate. Kuntz and Kauzmann  have argued that the intermolecular voids created on dehydrating a protein would lead to large unsatisfied intermolecular forces, which would provide a large driving force for rearranging exposed protein side chains and for the refolding of the polypeptide backbone in an attempt to reduce the size of the voids. Assuming a surface tension of 50 erg cm"2, the surface free energy for a spherical void of radius 5 A would be 22.5 kcal per mole of voids. Luscher-Mattli and Ruegg  also conclude that pronounced conformational changes occur at hydration levels below about 0.1 h, based on analysis of x-ray diffraction, infrared spectra, and thermodynamic data. Analysis of spin-spin interactions between electron spin resonance (ESR) spin probes covalently linked to lysozyme , however suggests that little or no change in protein conformation occurs down to a hydration level of about 0.02 h, at least to the resolution of the line shape analysis, which was 1 A. By contrast, laser Raman spectroscopic studies of lysozyme provide evidence for changes in the conformation of disulfide bridges, which are reordered (i.e., adopt their solution conformation) at a hydration level of 0.08 h, and for shifts in the position of a tryptophan side chain and the peptide backbone at hydration levels of 0.08-0.2 h , Circular dichroism spectra between 200 and 240 nm have been reported at different hydration levels for lysozyme and ribonuclease A . Comparison with spectra obtained in aqueous solution reveals differences in the relative contributions of bands at 210 and 220 nm, which may reflect changes in secondary structure at low hydration, although contributions from tryptophan side chains in this region of the spectrum are also expected and are known from Raman spectroscopic studies to undergo conformational changes on dehydration ,
Changes in the distribution of isotropic chemical shifts for lysozyme monitored by solid state ,3C cross-polarization magic-angle spinning (CP/MAS) NMR indicate that the distribution of conformational states becomes narrower as the protein is hydrated. These changes are observed for all peaks in the aliphatic region of the spectrum . A study of I3C CP/MAS NMR spectra for lysozyme as a function of hydration indicates that the change in the distribution of isotropic chemical shifts begins at a hydration level of 0.1-0.15 h and is largely complete at a hydration of 0.3-0.35 h . By contrast, in bovine serum albumin (BSA), enhance ments in spectral resolution on hydration are confined to the peak at 40 ppm. This begins at a hydration of about 0.2 h . The source of this behavior is not understood, but could reflect reordering of disulfide bridges, a decrease in the linewidth of contributing resonances from lysine side chains due to increased motional averaging on hydration, or titration shifts induced by hydration. The latter two explanations, however, seem unlikely. Proton-carbon cross-relaxation times do not change very significantly on hydration, while titration shifts would be expected to occur at much lower hydrations than is observed as water adds to ioniz-able side chains and normalizes their pKa values. With the exception of the changes in the peak at 40 ppm, hydration appears to have little effect on the solid state spectrum, and so the distribution of conformations sampled by fully hydrated BSA is as broad as that observed in the dry protein. The most interesting result of these studies, however, is that no significant changes are observed in either the rotating frame proton spin lattice relaxation time or cross-relaxation time on hydration. Therefore, the strength of dipolar coupling is not significantly affected by hydration of the protein. The preservation of efficient dipolar coupling suggests that the conformational changes that do occur on hydration do not lead to conformational states with any major expansion of free volume or weakened secondary interactions, which would significantly increase the reorientational freedom of protein groups .
B. An X-Ray Diffraction Study of a Dehydrated Protein
A recent x-ray diffraction study of dehydrated lysozyme provides the first detailed picture of the structure of a protein at low hydration levels. Protein crystals have a high solvent content (between 30 and 80% by volume) and tend to be rather soft and easily disordered. Dehydration of protein crystals usually leads to a loss of the diffraction pattern, due to intermolecular repositioning of individual protein molecules (lattice disorder). Kachalova et al. , however, found that triclinic crystals of hen egg white lysozyme cross-linked with glutaraldehyde retain their ability to diffract to high resolution even with a water content as low as 36 moles of water per mole of protein, the hydration level obtained at a relative humidity of 0.01. Dehydration leads to shifts in the relative positions of domains as well as numerous small displacements in the positions of individual atoms. Overall, drying leads to a contraction of the protein molecule. The molecular volume decreases from 1.93 X 104 Á3 for the fully hydrated protein to 1.82 X 104 Á3 for the "dry" protein, which represents an increase in the average packing density of 4—6%. The extent of displacements for main-chain atoms is comparable with the rms deviations calculated from the Debye-Waller factors for the dry protein, although there is no correlation between these parameters along the polypeptide chain. The largest dehydration-induced displacements (1.0-1.5 Á) are found at the C terminus, while values for the rest of the polypeptide backbone range from 0.2 to 0.9 Á. The av erage deviation for main-chain atoms is 0.6 A, and for all atoms is 0.9 A. The conformational changes are therefore small. The extent to which these results reflect the conformation of dehydrated or lyophilized powders of lysozyme is difficult to judge. It is possible that covalent cross-linking as well as lattice interactions prevented any large-scale conformational rearrangements on dehydration.
C. FTIR Studies of Dehydration-Induced Conformational Transitions
Prestrelski et al.  have measured Fourier-transform infrared spectra of a number of lyophilized proteins (basic fibroblast growth factor (basic FGF), ^-interferon, granulocyte-colony stimulating factor (G-CSF), a-lactalbumin, lysozyme, a-casein, and lactate dehydrogenase) as well as poly (L-lysine) using KBr pellets and attenuated total reflectance techniques. Comparison of the second-derivative spectra of the amide I band of the dry proteins with those obtained for proteins in aqueous solution indicate that dehydration causes significant pertuba-tion of the amide I region of the spectrum in many cases. Although Careri et al. observed changes in the amide I band of lysozyme on dehydration, these were attributed entirely to the removal of water without conformational change [3,10]. A comparison of the amide I band of dry and hydrated poly (L-lysine), however, suggests that the predominant source of changes in the second-derivative spectrum on dehydration is related to conformational changes rather than to removal of solvent , The preferred secondary structure for poly (L-lysine) in the dry state is the 3-sheet. When solutions of poly (L-lysine) in unordered or a-helix conformations are dried, the amide I band reveals the dehydration-induced transition to the (3-sheet structure. Apart from small frequency shifts, however, no change is observed in the amide I band when a solution of poly (L-lysine) prepared in a [3-sheet conformation is dried (i.e., there is little change in the amide I band that can be attributed to solvent effects on the carbonyl stretching mode). The changes in second-derivative amide I spectra on dehydration are quite variable and protein dependent. For example, little change is observed in the spectrum of G-CSF on dehydration, while the spectrum of casein is greatly altered on dehydration, with changes characteristic of a transition from a protein with little secondary structure to one with a large fraction of 3-sheet, similar to the changes observed for poly (L-lysine). In general, the resolved peaks in the second-derivative spectrum become broader on dehydration, indicative of the same type of static disorder observed by solid state 13C NMR spectroscopy. In some cases, the conformational changes were irreversible. For example, lyophilized casein is essentially insoluble in water, and lactate dehydrogenase is completely inactive on rehydration.
It is well known that polyols and sugars such as sucrose stabilize proteins against denaturation in solution (see Chapters 12 and 13). A similar protective effect is observed when proteins are lyophilized in the presence of sugars [4,64], In frared spectra of proteins lyophilized in the presence of such additives are very similar to the spectra observed in aqueous solution, indicating that these additives preserve the solution structure of the proteins during dehydration , These additives were also found to inhibit the conformational transitions observed with poly (L-lysine) on dehydration ,
Dehydration-induced conformational changes appear to be driven by the need to compensate for the loss of hydrogen bonding to water on dehydration [4,64], This is consistent with the transitions observed in poly (L-lysine) dehydration to a conformational state ((3-sheet structure) with a lower degree of solvation and a higher degree of intermolecular hydrogen bonding relative to an a-helix or unordered structure. Additives such as sucrose interact directly with the protein, and their hydroxyl groups could serve as water substitutes, forming hydrogen bonds with protein groups that would otherwise be unsatisfied in the dry state , Taken together, these results suggest that conformational changes do indeed occur as the protein is dehydrated. The extent of the changes that are induced can be quite variable and differ from protein to protein. In some proteins, little or no change in conformation occurs; in others, there is major rearrangement of the polypeptide backbone. Dehydration leads to a great reduction in conformational flexibility, and we suspect that the extent of dehydration-induced conformational changes will depend in part on where along the hydration isotherm these changes occur relative to the rigid-to-flexible transition (see also Sec. VII.E).
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