Is there an advantageous arrangement of aromatic residues in proteins? Statistical analysis of aromatic interactions in globular proteins

ABSTRACT An enhanced stability of enzymes in organic solvents is desirable under industrial conditions. The potential of lipases as biocatalysts is mainly limited by their denaturation in polar alcohols. In this study, we focused on selected solvent tunnels in lipase from Geobacillus stearothermophilus T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. The chemical and structural characteristics of each solvent tunnel were systematically analyzed. Selected residues were replaced with Phe, Tyr, or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed an enhanced stability up to 81-fold compared with that of the wild type. All stabilizing mutations were found in the longest tunnel detected in the “closed-lid” X-ray structure. The combination of Phe substitutions in an A187F/L360F double mutant resulted in an increase in unfolding temperature (Tm) of 7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with p-nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (kcat), though their stability properties mostly determined the transesterification capability. Seven crystal structures of different variants were solved, disclosing new π-π or CH/π intramolecular interactions and emphasizing the significance of aromatic interactions for improved solvent stability. This rational approach could be implemented for the stabilization of other enzymes in organic solvents. IMPORTANCE Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields; however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability, and we were inspired by this to redesign enzyme voids. The rational protein engineering of solvent tunnels of lipase from Geobacillus stearothermophilus is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. A structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting the solvent channel void.

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Role of long-range aromatic clique and community in protein stability

10.1101/319160 ◽

2018 ◽

Author(s):

P Mahanta ◽

A Bhardwaj ◽

VS Reddy ◽

S Ramakumar

Keyword(s):

Protein Stability ◽

Long Range ◽

Triosephosphate Isomerase ◽

Solvent Accessible Surface Area ◽

Aromatic Residues ◽

Aromatic Interactions ◽

The Stability ◽

Gh10 Xylanase ◽

Aromatic Interaction

AbstractAromatic interactions make an important contribution to protein structure, function, folding and have attracted intense study. Earlier studies on a recombinant xylanase from Bacillus sp. NG-27 (RBSX), which has the ubiquitous (beta/alpha)8-triosephosphate isomerase barrel fold showed that three aromatic residues to alanine substitutions, in the N-terminal and C-terminal regions, significantly decreased the stability of the enzyme. Of these mutations, F4A mutation decreased the stability of the enzyme by ∼4 degree C, whereas W6A mutation and Y343A mutation remarkably decreased the stability of the enzyme by ∼10 degree C. On the other hand, the F4W mutation did not affect the thermal stability of RBSX. We provide here a network perspective of aromatic-aromatic interactions in terms of aromatic clique community and long-range association. Our study reveals that disruption of long-range k-clique aromatic interaction cluster holding the N- and C-terminal regions are associated with the decreased stability of the enzyme. The present work reiterates as well as expands on those findings concerning the role of interactions between the N- and C-terminus in protein stability. Furthermore, comparative analyses of crystal structures of homologous pairs of proteins from thermophilic and mesophilic organisms emphasize the prevalence of long-range k-clique communities of aromatic interaction that may be playing an important role and highlights an additional source of stability in thermophilic proteins. The design principle based on clustering of long-range aromatic residues in the form of aromatic-clique and clique community may be effectively applied to enhance the stability of enzymes for biotechnological applications.DatabaseThe coordinates o fF4A, F4W, W6A, and Y343A are deposited in the PDB database under the accession numbers 5EFF, 5E58, 5EFD, and 5EBA respectively.AbbreviationsBSX, xylanase from Bacilllus sp. NG-27; RBSX, recombinant BSX xylanase; TIM, Triosephosphate isomerase; GH10, Glycosyl hydrolase family 10; 3D, three-dimensional; r.m.s.d, root mean square deviation; RSA, relative solvent accessible surface area; Tm, melting temperature; CD, Circular Dichroism; BHX, GH10 xylanase from Bacillus halodurans; BFX, GH10 xylanase from Bacillus firmus; TmxB, GH10 xylanase from Thermotoga maritima

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Statistical Analysis of the Relationship between the Folding Rate and Structure-Based Parameters of Globular Proteins

Seibutsu Butsuri ◽

10.2142/biophys.46.144 ◽

2006 ◽

Vol 46 (3) ◽

pp. 144-149

Author(s):

Kiyoto KAMAGATA ◽

Kunihiro KUWAJIMA

Keyword(s):

Statistical Analysis ◽

Globular Proteins ◽

Folding Rate ◽

The Relationship

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Statistical analysis of classification

Symposium - International Astronomical Union ◽

10.1017/s0074180900053420 ◽

1966 ◽

Vol 24 ◽

pp. 188-189

Author(s):

T. J. Deeming

Keyword(s):

Multivariate Analysis ◽

Statistical Analysis ◽

Classification Scheme ◽

Analytical Procedure ◽

Narrow Band ◽

Scientific Research ◽

Maximum Amount ◽

Amount Of Information

If we make a set of measurements, such as narrow-band or multicolour photo-electric measurements, which are designed to improve a scheme of classification, and in particular if they are designed to extend the number of dimensions of classification, i.e. the number of classification parameters, then some important problems of analytical procedure arise. First, it is important not to reproduce the errors of the classification scheme which we are trying to improve. Second, when trying to extend the number of dimensions of classification we have little or nothing with which to test the validity of the new parameters.Problems similar to these have occurred in other areas of scientific research (notably psychology and education) and the branch of Statistics called Multivariate Analysis has been developed to deal with them. The techniques of this subject are largely unknown to astronomers, but, if carefully applied, they should at the very least ensure that the astronomer gets the maximum amount of information out of his data and does not waste his time looking for information which is not there. More optimistically, these techniques are potentially capable of indicating the number of classification parameters necessary and giving specific formulas for computing them, as well as pinpointing those particular measurements which are most crucial for determining the classification parameters.

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Albumins as Embedding Media for Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100064025 ◽

1969 ◽

Vol 27 ◽

pp. 422-423 ◽

Cited By ~ 1

Author(s):

J. L. Farrant ◽

J. D. McLean

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

High Temperature ◽

Biological Material ◽

Globular Proteins ◽

The Past ◽

Antibody Staining ◽

Thin Sectioning ◽

Embedding Medium

For electron microscope techniques such as ferritin-labeled antibody staining it would be advantageous to have available a simple means of thin sectioning biological material without subjecting it to lipid solvents, impregnation with plastic monomers and their subsequent polymerization. With this aim in view we have re-examined the use of protein as an embedding medium. Gelatin which has been used in the past is not very satisfactory both because of its fibrous nature and the high temperature necessary to keep its solutions fluid. We have found that globular proteins such as the serum and egg albumins can be cross-linked so as to yield blocks which are suitable for ultrathin sectioning.

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Quantitative parallel EELS spectrum imaging developed as a new tool in AEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010013064x ◽

1992 ◽

Vol 50 (2) ◽

pp. 1202-1203

Author(s):

Gianluigi Botton ◽

Gilles L'espérance

Keyword(s):

Statistical Analysis ◽

Spatial Resolution ◽

Personal Computer ◽

Systematic Study ◽

Present Author ◽

Chemical Elements ◽

Detection Limits ◽

Research Groups ◽

Quantitative Performance ◽

Spectrum Imaging

As interest for parallel EELS spectrum imaging grows in laboratories equipped with commercial spectrometers, different approaches were used in recent years by a few research groups in the development of the technique of spectrum imaging as reported in the literature. Either by controlling, with a personal computer both the microsope and the spectrometer or using more powerful workstations interfaced to conventional multichannel analysers with commercially available programs to control the microscope and the spectrometer, spectrum images can now be obtained. Work on the limits of the technique, in terms of the quantitative performance was reported, however, by the present author where a systematic study of artifacts detection limits, statistical errors as a function of desired spatial resolution and range of chemical elements to be studied in a map was carried out The aim of the present paper is to show an application of quantitative parallel EELS spectrum imaging where statistical analysis is performed at each pixel and interpretation is carried out using criteria established from the statistical analysis and variations in composition are analyzed with the help of information retreived from t/γ maps so that artifacts are avoided.

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