scholarly journals High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phl p 2 As Revealed by X-Ray Crystallography

2009 ◽  
Vol 182 (4) ◽  
pp. 2141-2151 ◽  
Author(s):  
Sivaraman Padavattan ◽  
Sabine Flicker ◽  
Tilman Schirmer ◽  
Christoph Madritsch ◽  
Stefanie Randow ◽  
...  
Keyword(s):  
Conformational Epitope ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity

scholarly journals MBP-binding DARPins facilitate the crystallization of an MBP fusion protein

2018 ◽  
Vol 74 (9) ◽  
pp. 549-557 ◽  
Author(s):  
Rajesh Gumpena ◽  
George T. Lountos ◽  
David S. Waugh
Keyword(s):  
Fusion Protein ◽  
Catalytic Domain ◽  
Fusion Partner ◽  
High Quality ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Repeat Proteins ◽  
Dual Specificity ◽  
Ankyrin Repeat Proteins

The production of high-quality crystals is the main bottleneck in determining the structures of proteins using X-ray crystallography. In addition to being recognized as a very effective solubility-enhancing fusion partner,Escherichia colimaltose-binding protein (MBP) has also been successfully employed as a `fixed-arm' crystallization chaperone in more than 100 cases. Here, it is reported that designed ankyrin-repeat proteins (DARPins) that bind with high affinity to MBP can promote the crystallization of an MBP fusion protein when the fusion protein alone fails to produce diffraction-quality crystals. As a proof of principle, three different co-crystal structures of MBP fused to the catalytic domain of human dual-specificity phosphatase 1 in complex with DARPins are reported.

A New, Simple, High-Affinity Glycosidase Inhibitor:  Analysis of Binding through X-ray Crystallography, Mutagenesis, and Kinetic Analysis

2000 ◽  
Vol 122 (17) ◽  
pp. 4229-4230 ◽  
Author(s):  
Spencer J. Williams ◽  
Valerie Notenboom ◽  
Jacqueline Wicki ◽  
David R. Rose ◽  
Stephen G. Withers
Keyword(s):  
Kinetic Analysis ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Inhibitor Analysis ◽  
Glycosidase Inhibitor

scholarly journals Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

2010 ◽  
Vol 76 (19) ◽  
pp. 6351-6359 ◽  
Author(s):  
Toshihiko Kitajima ◽  
Yasunori Chiba ◽  
Yoshifumi Jigami
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Methylotrophic Yeast ◽  
Culture Conditions ◽  
Crystallographic Analysis ◽  
Gene Encoding ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Standard Culture ◽  
Resistant Mutants

ABSTRACT The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

Structure-Based Design, Synthesis, and X-ray Crystallography of a High-Affinity Antagonist of the Grb2-SH2 Domain Containing an Asparagine Mimetic

1999 ◽  
Vol 42 (13) ◽  
pp. 2358-2363 ◽  
Author(s):  
Pascal Furet ◽  
Carlos García-Echeverría ◽  
Brigitte Gay ◽  
Joseph Schoepfer ◽  
Martin Zeller ◽  
...  
Keyword(s):  
Sh2 Domain ◽  
Design Synthesis ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity

scholarly journals Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy

2005 ◽  
Vol 102 (50) ◽  
pp. 17958-17963 ◽  
Author(s):  
J. R. Simard ◽  
P. A. Zunszain ◽  
C.-E. Ha ◽  
J. S. Yang ◽  
N. V. Bhagavan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Nmr Spectroscopy ◽  
Binding Sites ◽  
Fatty Acid Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity

scholarly journals Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

2015 ◽  
Vol 112 (29) ◽  
pp. 9004-9009 ◽  
Author(s):  
Hemant Kumar ◽  
Janet S. Finer-Moore ◽  
H. Ronald Kaback ◽  
Robert M. Stroud
Keyword(s):  
Hydrophobic Interactions ◽  
Side Chain ◽  
Lactose Permease ◽  
Protonation Site ◽  
Conformationally Constrained ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Biochemical Studies ◽  
Trp Mutant

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.

Characterization of a high-affinity sialic acid-specific CBM40 from Clostridium perfringens and engineering of a divalent form

2016 ◽  
Vol 473 (14) ◽  
pp. 2109-2118 ◽  
Author(s):  
João P. Ribeiro ◽  
William Pau ◽  
Carlo Pifferi ◽  
Olivier Renaudet ◽  
Annabelle Varrot ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Clostridium Perfringens ◽  
Carbohydrate Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Carbohydrate Binding Modules ◽  
Dimeric Form ◽  
Calorimetric Studies

CBMs (carbohydrate-binding modules) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the CBM40 family, coded from a section of the gene NanI from Clostridium perfringens. Glycan arrays revealed its preference towards α(2,3)-linked sialosides, which was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)-sialyl-lactose with a Kd of ∼30 μM, the highest affinity value for this class of proteins. Inspired by lectins' structure and their arrangement as multimeric proteins, we have engineered a dimeric form of the CBM, and using SPR (surface plasmon resonance) we have observed 6–11-fold binding increases due to the avidity affect. The structures of the CBM, resolved by X-ray crystallography, in complex with α(2,3)- or α(2,6)-sialyl-lactose explain its binding specificity and unusually strong binding.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

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