Hydration-induced conformational and flexibility changes in lysozyme at low water content

349

311

Changes in the secondary structure of some dozen different proteins upon lyophilization of their aqueous solutions have been investigated by means of Fouriertransform infrared spectroscopy in the amide IHI band region. Dehydration markedly (but reversibly) alters the secondary structure of all the proteins studied, as revealed by both the quantitative analysis of the second derivative spectra and the Gaussian curve fitting of the original infrared spectra. Lyophilization substantially increases the ,8-sheet content and lowers the a-helix content of all proteins. In all but one case, proteins become more ordered upon lyophilization.Consider the common laboratory and bioindustrial process of lyophilization or freeze-drying of aqueous solutions of proteins. Suppose that this lyophilization is carried out such that no irreversible damage to the protein ensues-i.e., when the lyophilized protein is redissolved in water, it exhibits the same properties as prior to lyophilization. The question still remains whether the protein structure in the lyophilized form is native or whether the lyophilization has resulted in reversible protein denaturation. Apart from its biochemical interest, the answer has important biotechnological implications. For example, proteins that have been lyophilized (which is how research and pharmaceutical protein preparations are usually stored) undergo moisture-triggered aggregation (1). To understand the mechanism of this undesirable phenomenon and to develop rational strategies for its prevention, structural information on proteins in the solid-e.g., lyophilized-form is needed. In addition, lyophilized enzymes suspended in organic solvents have proven to be useful synthetic catalysts (2); enzyme structural data should help maximize their performance.The issue of protein conformation in the lyophilized form is controversial. For example, the results of Fourier-transform infrared (FTIR) spectroscopic investigations of hen egg-white lysozyme were interpreted to indicate that lysozyme structure in either aqueous solution or the lyophilized state is the same (3-6). This conclusion was supported by some hydrogen isotope-exchange studies (6, 7) but contradicted by others (8). Raman (9-11) and solid-state NMR (12) studies have suggested significant (reversible) structural changes occurring in lysozyme upon lyophilization. Likewise, recent hydrogen isotope-exchange/high-resolution NMR (13) and FTIR (14,15) investigations of various proteins strongly point to lyophilization-induced reversible denaturation.Recent advances, both instrumentational and conceptual, in FTIR spectroscopy make it a method of choice for examining the structure of solid proteins. This has been illustrated by the scholarly work of Prestrelski et al. (14,15), who have employed this methodology to quantify changes in the secondary structure of proteins caused by lyophilization. Using the second derivatives of the vibrational spectra of proteins in the amide I band region (1600-1720 cm-1), these authors have calculated so-call...

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confidence: 65%

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confidence: 99%

Lyophilization-induced reversible changes in the secondary structure of proteins.

Griebenow

Klibanov

1995

349

311

“…The comparison is of general interest because the protein powder is fully hydrated and is expected to have a structure corresponding to that in solution and to be biologically active (20)(21)(22). Use of a hydrated powder in the neutron scattering experiments yields better data than a solution study due to the higher concentration of the protein and the lower background from 2H20.…”

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confidence: 99%

Dynamics of myoglobin: comparison of simulation results with neutron scattering spectra.

Smith

Kuczera

Karplus

1990

156

Molecular dynamics simulations are used to calculate the incoherent neutron scattering spectra of myoglobin between 80 K and 325 K and compared with experimental data. There is good agreement over the entire temperature range for the elastic, quasi-elastic, and inelastic components of the scattering. This provides support for the accuracy of the simulations of the internal motions that make the dominant contributions to the atomic displacements on a time scale of 0.3-100 ps (100-0.3 cm-'). Analysis of the simulations shows that at low temperatures a harmonic description of the molecule is appropriate and that the molecule is trapped in localized regions of conformational space. At higher temperatures the scattering arises from a combination of vibrations within wells (substates) and transitions between them; the latter contribute to the quasi-elastic scattering.Experimental and theoretical studies ofthe internal dynamics of proteins can provide insight into the atomic interactions determining their structure and function (1). In heme proteins such as myoglobin, the internal motions have been shown to have an essential role in ligand binding (1-9). Two extreme models for the internal motions have been considered. In one the fluctuations are assumed to occur within a single multidimensional well that is harmonic or quasiharmonic (10). The other model assumes that there exist multiple minima or substates on the protein potential surface (2, 5, 11, 12); the internal motions correspond to a superposition of oscillations within the wells and transitions among them (11). Photolysis rebinding studies (2, 5, 12) and simulations (11) of myoglobin provide strong evidence for the substate model, which may be generally applicable to globular proteins (12). The simulations have shown that structures associated with different substates are similar but differ in detail; for myoglobin the differences can be described in terms of rigid body displacements of the helices with rearrangements of the connecting loops and reorientation of the side chains involved in helix contacts. At low temperatures individual protein molecules are trapped in local regions of conformational space and static disorder, corresponding to an inhomogeneous macroscopic system, results; at higher temperatures transitions between the substates, which involve the crossing of energy barriers, are possible. Such behavior is analogous to that found in other complex systems (13).Although much has been learned about protein motions from simulations, detailed experimental tests have been limited by the accessible time scales. Comparisons with x-ray, NMR, and fluorescence depolarization data have provided evidence that supports the simulation results (1), although it was suggested on the basis of the Mossbauer spectrum of myoglobin that the time scale of the simulated atomic fluctuations is two orders of magnitude too short (14); a reinterpretation of the results shows there is no inconsistency between the Mossbauer and simulation dynamics (15).Incoherent neu...

“…Changes in the structure and internal dynamics of proteins as a function of solvent conditions at physiological temperatures have been found by using several experimental techniques. In the absence of minimal hydration, lysozyme does not function (25), and related NMR measurements of exchangeability of the main chain amide hydrogens suggest a hydration-related increase in conformational flexibility of the protein (26), which may be coupled to (probably small) Raman-detected conformational changes that appear to be necessary before activity (26,27). Rayleigh scattering of Möss-bauer radiation demonstrated an increase of dynamic amplitudes on hydration of myoglobin (28).…”

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confidence: 99%

Solvent dependence of dynamic transitions in protein solutions

Réat

Dunn

Ferrand

et al. 2000

125

128

A transition as a function of increasing temperature from harmonic to anharmonic dynamics has been observed in globular proteins by using spectroscopic, scattering, and computer simulation techniques. We present here results of a dynamic neutron scattering analysis of the solvent dependence of the picosecond-time scale dynamic transition behavior of solutions of a simple single-subunit enzyme, xylanase. The protein is examined in powder form, in D 2O, and in four two-component perdeuterated single-phase cryosolvents in which it is active and stable. The scattering profiles of the mixed solvent systems in the absence of protein are also determined. The general features of the dynamic transition behavior of the protein solutions follow those of the solvents. The dynamic transition in all of the mixed cryosolvent-protein systems is much more gradual than in pure D 2O, consistent with a distribution of energy barriers. The differences between the dynamic behaviors of the various cryosolvent protein solutions themselves are remarkably small. The results are consistent with a picture in which the picosecond-time scale atomic dynamics respond strongly to melting of pure water solvent but are relatively invariant in cryosolvents of differing compositions and melting points. X-ray diffraction, dynamic neutron scattering, and various spectroscopies have demonstrated a quantitative change in the nature of internal motions of proteins, at Ϸ200-220 K (1-8). Below this transition, the internal motions are essentially harmonic whereas above it anharmonic dynamics contribute and, at physiological temperatures, dominate the internal fluctuations. The anharmonic motions may involve confined continuous diffusion (6, 9) and͞or jump diffusion between potential energy wells associated with ''conformational substates'' of slightly different structure in which proteins are trapped below the transition (2, 10-12).Correlations have been made between some protein functions (such as ligand binding, electron transfer, and proton pumping) and the presence of equilibrium anharmonic motion (8,(13)(14)(15)(16)(17). Recent x-ray diffraction work on carbon monoxy-myoglobin showed that, below 180 K, photodissociated ligands migrate to specific sites within an internal cavity of an essentially immobilized, frozen protein, from which they subsequently rebind by thermally activated barrier crossing (18). On photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels.The increased flexibility conferred by anharmonic dynamics may indeed be required for some proteins to rearrange their structures to achieve functional configurations. However, the forms and time scales of the anharmonic motions required for function are in general unknown. In particular, it was recently shown that, in cases of enzyme activity in a cryosolvent, the rate-limiting step is independent of the Ϸ220 K dynamic transition (19,20). Furthermore, it was demonstrated that dynamic transition temperatures in a ...