Cross‐Linked Enzyme Crystals (CLECs): Efficient and Stable Biocatalysts for Preparative Organic Chemistry

Zelinski, Thomas; Waldmann, Herbert

doi:10.1002/anie.199707221

Cited by 68 publications

(41 citation statements)

References 17 publications

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“…Although, crosslinked enzyme crystals or CLEC's 26 have important practical applications, there have been few detailed structural studies of the effects of crosslinking. The present studies show that, using limited crosslinking, it is possible to stabilize protein crystals in the presence of denaturants without significantly affecting the diffraction limit.…”

Section: Discussionmentioning

confidence: 99%

“…Crosslinked enzyme crystals (CLECs) have tremendous potential as biocatalysts under harsh conditions. 26 In some cases strong crosslinking has been shown to physically strengthen the crystal to mechanical damage 40,56 without major changes in the diffraction of the protein. RNase S is stabilized by mild glutaraldehyde crosslinking but loses its diffraction pattern after crosslinking for extended periods ( Fig.…”

Section: Glutaraldehyde Crosslinking Does Not Affect the Protein Strumentioning

confidence: 99%

“…Substrates as large as 3,000 D have been shown to diffuse through channels (25 Å diameter) in crosslinked thermolysin. 25,26 The primary difficulty in using crystallography to examine protein structures in the presence of denaturants is that protein crystals often dissolve or crack at concentrations of denaturant (or low pH) well before the Cm of the protein in solution. Therefore studies of protein crystals in denaturants have either been carried out using low denaturant concentrations or else with protein crystals such as those of tetragonal hen egg-white lysozyme which are relatively resistant to high denaturant concentrations.…”

Section: Introductionmentioning

confidence: 99%

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X‐ray crystallographic studies of the denaturation of ribonuclease S

Ratnaparkhi

Varadarajan

1999

Proteins

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In an attempt to view the onset of urea denaturation in ribonuclease we have collected X-ray diffraction data on ribonuclease S crystals soaked in 0, 1.5, 2, 3, and 5 molar urea. At concentrations above 2 M urea, crystals were stabilized by glutaraldehyde crosslinking. We have also collected data on ribonuclease S crystals at low pH in an attempt to study the onset of pH denaturation. The resolution of the datasets range from 1.9 to 3.0 Å . Analysis of the structures reveals an increase in disorder with increasing urea concentration. In the 5 M urea structure, this increase in disorder is apparent all over the structure but is larger in loop and helical regions than in the ␤ strands. The low pH structure shows a very similar pattern of increased disorder. In addition there is a major change in the position of the main chain (G 1 Å ) in the 65-72 turn region. This region has previously been shown to be involved in one of the initial steps of unfolding in the reduction of ribonuclease A. Crystallographic analyses in the presence of denaturant, when combined with controlled crosslinking, can thus provide detailed structural information that is related to the initial steps of unfolding in solution. Proteins 1999;36:282-294.

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Section: Discussionmentioning

confidence: 99%

Section: Glutaraldehyde Crosslinking Does Not Affect the Protein Strumentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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X‐ray crystallographic studies of the denaturation of ribonuclease S

Ratnaparkhi

Varadarajan

1999

Proteins

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“…Osmolyte Does Not Perturb the Crystal Structure of RNase S-The crystalline state of a protein has large empty channels, which allow molecules as large as 3000 Da to diffuse in the crystal lattice and interact with the protein (37,38). Osmolytes should therefore diffuse into the crystal lattice and be in a position to bind protein molecules inside the crystal lattice.…”

Section: Effect Of Sarcosine On the Stokes Radius Of S Pep And S Pro-mentioning

confidence: 99%

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Ratnaparkhi¹,

Varadarajan²

2001

Journal of Biological Chemistry

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Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein⅐peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 M sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4°C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 M sarcosine increases with temperature, ranging from ؊0.52 kcal mol ؊1 at 20°C to ؊5.4 kcal mol ؊1 at 60°C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.Osmolytes are molecules used in nature to protect organisms against stresses of high osmotic pressure. These compounds have also been found to stabilize the native state of proteins relative to the unfolded state. The mechanism of this stabilization is not completely understood (1-4), although it is believed to result primarily from an unfavorable free energy of interaction between the osmolyte and the unfolded state of the protein (5, 6). Proteins retain activity in the presence of osmolytes suggesting that native state structure and dynamics are not greatly perturbed. However, there is little high resolution structural information available for proteins in the presence of osmolytes. The main classes of osmolytes are sugars, methyl ammonium derivatives, polyhydric alcohols, and amino acids and their derivatives (1). Molar concentrations of all the above classes of molecules have been shown to stabilize proteins. Many organisms accumulate osmolytes under conditions of water stress, such as high salinity, desiccation or freezing. In vivo, amino acid derivatives also counteract the accumulation of urea, which is capable of denaturing proteins. Marine cartilaginous fishes use, as osmolytes, a combination of urea and methylamines, i.e. a denaturant and a stabilizer, in a 2:1 ratio (1,7,8). Stability studies on RNase T1, RNase A, and other proteins have shown that, if the 2:1 ratio of urea:methylamine is maintained, the methylamine is able to counteract the destabilizing effect of the denaturant (1,7,8).In an effort to further our understanding of the basis of osmolyte stabilization of proteins, we have studied the stabilization of the fragment complementation system ribonuclease S (RNase S) 1 in four structurally similar osmolytes, namely sarcosine, betaine, trimethylamine-N-oxide (TMAO), and taurine. RNase S is a complex of two fragm...

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“…Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for cytochemistry and electron microscopy (Sabatini et al, 1963), immobilization of enzymes and whole cells (Quiocho and Richards, 1966;Schejter and Bareli, 1970;Walt and Agayn, 1994;Khalaf et al, 1996;Lalonde et al, 1997), and protein crystal stabilization for X-ray crystallography (Wang et al, 1998;Lusty, 1999;Hall et al, 2005;Heras and Martin, 2005;Iimura et al, 2005) as well as for biocatalysis (Margolin, 1996;Zelinski and Waldmann, 1997;Haring and Schreier, 1999;Leisola et al, 2001;Margolin and Navia, 2001;Iimura et al, 2005;Roy and Abraham, 2006). Inspite of its wide applicability and high efficiency as a crosslinking agent, the mechanism and chemistry involved in this process is not fully understood (Whipple and Ruta, 1974;Walt and Agayn, 1994).…”

Section: Introductionmentioning

confidence: 99%

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X‐ray structure analysis

Wine

Cohen-Hadar

Freeman

et al. 2007

Biotech & Bioengineering

178

130

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Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.

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Cross‐Linked Enzyme Crystals (CLECs): Efficient and Stable Biocatalysts for Preparative Organic Chemistry

Cited by 68 publications

References 17 publications

X‐ray crystallographic studies of the denaturation of ribonuclease S

X‐ray crystallographic studies of the denaturation of ribonuclease S

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X‐ray structure analysis

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