Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions

Schinkel, Jeffrey E.; Downer, Nancy W.; Rupley, John A.

doi:10.1021/bi00323a018

Cited by 79 publications

(51 citation statements)

References 23 publications

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“…Hydration-induced mobility in a solid protein was demonstrated previously in lysozyme powder using the hydrogen exchange reaction (Schinkel et al, 1985). At the final stage of hydration, where the motion is most prominent, the degree of hydration is close to that in SSI crystals (41 vol%, Satow et al, 1973).…”

Section: Powdercsupporting

confidence: 60%

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state

et al. 1996

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Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core.The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10"'s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 X lo-* s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (> 10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between IO-' and lO"'s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 7 0 and/or 73 are likely to have small-amplitude motions.Keywords: deuterium NMR; methionyl side chain dynamics; hydration of solid proteins; protein internal motion; specific deuteration; Streptomyces subtilisin inhibitor

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Section: Powdercsupporting

confidence: 60%

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state

et al. 1996

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“…Such protection has been observed with lyophilized lysozyme powders. 23 The H-D exchange is further decreased with an increasing percentage of trehalose (Table II). This suggests that 20% trehalose (1 : 4 weight ratio of trehalose/protein) is insufficient to protect exchangeable amide protons and an increasing trehalose content (up to 1 : 1) increases the protection.…”

Section: Extent Of Solvent-exposed Protons Of Trehalose and Proteinsmentioning

confidence: 94%

Fourier transform infared spectroscopy investigation of protein conformation in spray‐dried protein/trehalose powders

French

Arakawa

2004

Biopolymers

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Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure. Trehalose reduced the magnitude of the changes in beta sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns). Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.

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“…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)(4)(5)(6). This conclusion was supported by some hydrogen isotope-exchange studies (6,7) but contradicted by others (8). Raman (9)(10)(11) and solid-state NMR (12) studies have suggested significant (reversible) structural changes occurring in lysozyme upon lyophilization.…”

mentioning

confidence: 99%

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

Griebenow

Klibanov

1995

Proc. Natl. Acad. Sci. U.S.A.

349

311

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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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Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions

Cited by 79 publications

References 23 publications

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state

Fourier transform infared spectroscopy investigation of protein conformation in spray‐dried protein/trehalose powders

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

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