scholarly journals Designing better diffracting crystals of biotin carboxyl carrier protein fromPyrococcus horikoshiiby a mutation based on the crystal-packing propensity of amino acids

2017 ◽  
Vol 73 (9) ◽  
pp. 757-766 ◽  
Author(s):  
Kazunori D. Yamada ◽  
Naoki Kunishima ◽  
Yoshinori Matsuura ◽  
Koshiro Nakai ◽  
Hisashi Naitow ◽  
...  
Keyword(s):  
Amino Acids ◽  
Crystal Packing ◽  
Protein Crystallization ◽  
Single Site ◽  
Rational Approach ◽  
Carrier Protein ◽  
Structural Class ◽  
Biotin Carboxyl Carrier Protein ◽  
Crystallization Experiment ◽  
Β Sheet

An alternative rational approach to improve protein crystals by using single-site mutation of surface residues is proposed based on the results of a statistical analysis using a compiled data set of 918 independent crystal structures, thereby reflecting not only the entropic effect but also other effects upon protein crystallization. This analysis reveals a clear difference in the crystal-packing propensity of amino acids depending on the secondary-structural class. To verify this result, a systematic crystallization experiment was performed with the biotin carboxyl carrier protein fromPyrococcus horikoshiiOT3 (PhBCCP). Six single-site mutations were examined: Ala138 on the surface of a β-sheet was mutated to Ile, Tyr, Arg, Gln, Val and Lys. In agreement with prediction, it was observed that the two mutants (A138I and A138Y) harbouring the residues with the highest crystal-packing propensities for β-sheet at position 138 provided better crystallization scores relative to those of other constructs, including the wild type, and that the crystal-packing propensity for β-sheet provided the best correlation with the ratio of obtaining crystals. Two new crystal forms of these mutants were obtained that diffracted to high resolution, generating novel packing interfaces with the mutated residues (Ile/Tyr). The mutations introduced did not affect the overall structures, indicating that a β-sheet can accommodate a successful mutation if it is carefully selected so as to avoid intramolecular steric hindrance. A significant negative correlation between the ratio of obtaining amorphous precipitate and the crystal-packing propensity was also found.

scholarly journals Structure of a monoclinic polymorph of human carbonic anhydrase II with a doubledaaxis

2010 ◽  
Vol 66 (5) ◽  
pp. 628-634 ◽  
Author(s):  
Arthur H. Robbins ◽  
John F. Domsic ◽  
Mavis Agbandje-McKenna ◽  
Robert McKenna
Keyword(s):  
Carbonic Anhydrase ◽  
Crystal Packing ◽  
Rotation Axis ◽  
Unit Cell ◽  
Carbonic Anhydrase Ii ◽  
Monoclinic Unit Cell ◽  
Structure Solution ◽  
Unit Cells ◽  
Human Carbonic Anhydrase ◽  
Β Sheet

The crystal structure of human carbonic anhydrase II with a doubledaaxis from that of the usually observed monoclinic unit cell has been determined and refined to 1.4 Å resolution. The diffraction data withh= 2n+ 1 were systematically weaker than those withh= 2n. Consequently, the scaling of the data, structure solution and refinement were challenging. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of ½ alonga, but one of the molecules has two alternate positions related by a rotation of approximately 2°. This rotation axis is located near the edge of the central β-sheet, causing a maximum distance disparity of 1.7 Å between equivalent atoms on the diametrically opposite side of the molecule. The crystal-packing contacts are similar to two sequential combined unit cells alongaof the previously determined monoclinic unit cell. Abnormally high finalRcrystandRfreevalues (20.2% and 23.7%, respectively) are not unusual for structures containing pseudo-translational symmetry and probably result from poor signal to noise in the weakh-odd data.

scholarly journals TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution

2005 ◽  
Vol 387 (2) ◽  
pp. 401-409 ◽  
Author(s):  
Jolanta KOPEC ◽  
Alexander BERGMANN ◽  
Gerhard FRITZ ◽  
Elisabeth GROHMANN ◽  
Walter KELLER
Keyword(s):  
Amino Acids ◽  
Broad Host Range ◽  
Mobility Shift ◽  
Gram Positive ◽  
Nick Site ◽  
Α Helix ◽  
Electrophoretic Mobility Shift Assays ◽  
Dna Complex ◽  
Β Sheet

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein–DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 °C. CD spectra measured at 20 °C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase–oriT complex.

scholarly journals Surface Engineering of Top7 to Facilitate Structure Determination

2022 ◽  
Vol 23 (2) ◽  
pp. 701
Author(s):  
Yuki Ito ◽  
Takuya Araki ◽  
Shota Shiga ◽  
Hiroyuki Konno ◽  
Koki Makabe
Keyword(s):  
Structure Determination ◽  
Surface Engineering ◽  
De Novo ◽  
Crystal Packing ◽  
Structure Model ◽  
X Ray ◽  
Hexahistidine Tag ◽  
Model Protein ◽  
Β Sheet ◽  
Packing Interactions

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

scholarly journals Protein crystallization in the presence of amino acids and their derivatives. (2) The mechanism

2008 ◽  
Vol 64 (a1) ◽  
pp. C581-C581
Author(s):  
L. Ito ◽  
G. Sazaki ◽  
K. Shiraki ◽  
T. Shibano ◽  
A. Matsuoka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Crystallization

Effect of the weather conditions during solution preparation on lysozyme crystallization

2017 ◽  
Vol 50 (5) ◽  
pp. 1341-1351 ◽  
Author(s):  
Qing-Di Cheng ◽  
Rui-Qing Chen ◽  
Jin He ◽  
Da-Wei Li ◽  
Fan Yang ◽  
...  
Keyword(s):  
Particulate Matter ◽  
Environmental Factors ◽  
Protein Crystallization ◽  
Weather Conditions ◽  
Crystallization Experiment ◽  
Solution Preparation ◽  
Crystallization Solution ◽  
Potential Factor ◽  
Weather Parameters ◽  
Set Up

Protein crystallization is a delicate process that is always sensitive to environmental factors. When the environmental factors are not well controlled or not controlled at all, identical crystallization droplets from the same mother liquid may yield different crystallization results. One environmental factor, the weather conditions during crystallization solution preparation, is not usually considered as a parameter for protein crystallization. In this paper, it is shown that the weather parameters during preparation of the crystallization experiment, including the ambient temperature, humidity, pressure and particulate matter in the air, can all affect the reproducibility of lysozyme crystallization. An identical lysozyme crystallization experiment was repeated for an entire year, and the weather conditions when each crystallization experiment was set up were recorded along with the crystallization results. Among the parameters recorded, the humidity during the experiment setup showed the strongest effect on lysozyme crystallization. On the basis of these results, it is suggested that the weather conditions during crystallization solution preparation should be considered as a potential factor that can influence protein crystallization.

scholarly journals Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes

2005 ◽  
Vol 61 (6) ◽  
pp. 750-754 ◽  
Author(s):  
Vincenzo Granata ◽  
Nicholas G. Housden ◽  
Stephen Harrison ◽  
Colette Jolivet-Reynaud ◽  
Michael G. Gore ◽  
...  
Keyword(s):  
Crystal Packing ◽  
Single Site ◽  
Mutant Protein ◽  
Protein L

Designed Amino Acids That Induce β-Sheet Folding and β-Sheet Interactions in Peptides

2001 ◽  
pp. 572-574
Author(s):  
James S. Nowick ◽  
Kit S. Lam ◽  
Chris M. Gothard ◽  
Jeffrey K. Huon ◽  
William E. Kemnitzer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Β Sheet
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