Effective Improvement of D-Phenylglycine Aminotransferase Solubility by Protein Crystal Contact Engineering

2012 ◽  
Vol 22 (3) ◽  
pp. 147-155 ◽  
Author(s):  
Aiya Chantarasiri ◽  
Vithaya Meevootisom ◽  
Duangnate Isarangkul ◽  
Suthep Wiyakrutta
Keyword(s):  
Protein Crystal ◽  
Contact Engineering ◽  
Crystal Contact

scholarly journals Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases

Crystals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 975
Author(s):  
Brigitte Walla ◽  
Daniel Bischoff ◽  
Robert Janowski ◽  
Nikolas von den Eichen ◽  
Dierk Niessing ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Crystal Packing ◽  
Protein Crystallization ◽  
Lactobacillus Brevis ◽  
Protein Crystal ◽  
Wild Type ◽  
Alcohol Dehydrogenases ◽  
Crystallization Conditions ◽  
Contact Engineering ◽  
Crystal Contact

Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E > Q126K > LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies.

scholarly journals Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study

2020 ◽  
Vol 1864 (2) ◽  
pp. 129418 ◽  
Author(s):  
Siqin Bala ◽  
Shoko Shinya ◽  
Arpita Srivastava ◽  
Marie Ishikawa ◽  
Atsushi Shimada ◽  
...  
Keyword(s):  
Protein Crystal ◽  
Flexible Loop ◽  
Contact Free ◽  
Crystal Contact

Rational Crystal Contact Engineering ofLactobacillus brevisAlcohol Dehydrogenase To Promote Technical Protein Crystallization

2019 ◽  
Vol 19 (4) ◽  
pp. 2380-2387 ◽  
Author(s):  
Phillip Nowotny ◽  
Johannes Hermann ◽  
Jianing Li ◽  
Angela Krautenbacher ◽  
Kai Klöpfer ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Contact Engineering ◽  
Crystal Contact

scholarly journals Intentional crystal-contact-free space in protein crystal

2014 ◽  
Vol 70 (a1) ◽  
pp. C339-C339
Author(s):  
Rei Matsuoka ◽  
Yasuaki Komuro ◽  
Yuji Sugita ◽  
Daisuke Kohda
Keyword(s):  
Electron Density ◽  
Free Space ◽  
Protein Import ◽  
Model Building ◽  
Protein Complexes ◽  
Dynamics Simulation ◽  
Protein Crystal ◽  
Α Helix ◽  
Contact Free ◽  
Crystal Contact

To understand the function of proteins, it is essential to perform the structural analysis of the protein complexes with ligands, such as substrates or partner molecules. The motions of ligands are restricted by the contacts with neighbor protein molecules in the crystal lattice. Here, we propose a new technique to analyze dynamics of a ligand in the bound state preserved in the crystal-contact-free space, which is intentionally created in protein crystals. We used Tom20 as a target protein. Tom20 functions as a general protein import receptor, by recognizing N-terminal signal sequences (presequences) of mitochondrial matrix proteins. Our working hypothesis is that the promiscuous specificity of Tom20 is attributed to the large mobility of the presequneces in the binding groove of Tom20 (1,2). Our aim is to obtain electron density that reflects the large mobility of a presequence in the crystal-contact-free space. In order to create the crystal-contact-free space, we took advantage of a protein fused with maltose binding protein (MBP). The key of the design is the connection of the two proteins firmly. We fused the C-terminal α-helix of MBP and the N-terminal α-helix of Tom20 seamlessly. After a systematic model building study, we decided to use a design with four residues inserted in the linker region. We found smeared electron density in the binding site of presequences in the difference Fourier electron-density map. We attached an iodine atom at the N-terminus of the presequence and confirmed the N-terminal position in the smeared electron density. We performed molecular dynamics simulation without the tethering in solution (3). The electron density simulated from the MD trajectory was fully consistent with the smeared electron density in the crystal contact-free space. We concluded that the smeared electron density corresponded to the partially overlapping region of the multiple states of the bound presequence.

scholarly journals Crystal Contact Engineering Enables Efficient Capture and Purification of an Oxidoreductase by Technical Crystallization

2020 ◽  
Vol 15 (11) ◽  
pp. 2000010
Author(s):  
Phillip Grob ◽  
Max Huber ◽  
Brigitte Walla ◽  
Johannes Hermann ◽  
Robert Janowski ◽  
...  
Keyword(s):  
Contact Engineering ◽  
Crystal Contact

Spot-scan imaging of ice-embedded catalase crystal on a slow-scan CCD camera in a 400-kV electron cryomicroscope

1994 ◽  
Vol 52 ◽  
pp. 98-99
Author(s):  
Wah Chiu ◽  
Michael Sherman ◽  
Jaap Brink
Keyword(s):  
Electron Diffraction ◽  
Ccd Camera ◽  
Protein Crystal ◽  
Modulation Transfer ◽  
Measured Intensity ◽  
3 Dimensional ◽  
Dimensional Reconstruction ◽  
Diffraction Patterns ◽  
Complex Crystal

In protein electron crystallography, both low dose electron diffraction patterns and images are needed to provide accurate amplitudes and phases respectively for a 3-dimensional reconstruction. We have demonstrated that the Gatan 1024x1024 model 679 slow-scan CCD camera is useful to record electron diffraction intensities of glucose-embedded crotoxin complex crystal to 3 Å resolution. The quality of the electron diffraction intensities is high on the basis of the measured intensity equivalence ofthe Friedel-related reflections. Moreover, the number of patterns recorded from a single crystal can be as high as 120 under the constraints of radiation damage and electron statistics for the reflections in each pattern.A limitation of the slow-scan CCD camera for recording electron images of protein crystal arises from the relatively large pixel size, i.e. 24 μm (provided by Gatan). The modulation transfer function of our camera with a P43 scintillator has been determined for 400 keV electrons and shows an amplitude fall-off to 0.25 at 1/60 μm−1.

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