Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Kim, Heung-Bok; Webster, Cecelia; Roberts, Justin K. M.; Kositsawat, Juthamas; Hung, Li Wei; Terwilliger, Thomas C.; Kim, Chang-Yub

doi:10.1007/s10969-012-9124-8

Cited by 5 publications

(9 citation statements)

References 43 publications

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“…Furthermore, these ligands are able to interact through different elementary interactions and with different intensities with proteins, revealing remarkable specificity for some of them (Clonis et al ., ). Thiacarbocyanines (Boto et al ., , ) and Cibacron Blue F3G‐A (Subramanian, ; Kim et al ., ) are examples of dyes with significant application in affinity chromatography.…”

Section: Introductionmentioning

confidence: 99%

Protein purification by aminosquarylium cyanine dye‐affinity chromatography

Silva

Graça

Reis

et al. 2013

Biomedical Chromatography

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The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.

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Section: Introductionmentioning

confidence: 99%

Protein purification by aminosquarylium cyanine dye‐affinity chromatography

Silva

Graça

Reis

et al. 2013

Biomedical Chromatography

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“…We then used an independent experimental method to test if we can get validation for any proteins predicted by our computational method. A high-throughput method of using Cibacron Blue F3GA dye-ligand affinity chromatography (DLAC) described previously was carried out 40 , 41 . In this method, initially we used M.tb cell extract proteins to bind on the dye and elute with nucleotide ligands after intensive washing, and the proteins eluted by nucleotides were analyzed by 2D-gel analysis and mass spectroscopy (MS) for identification of each eluted protein shown on 2D-gel.…”

Section: Resultsmentioning

confidence: 99%

“…According to these purified proteins data, some part of NTPome data were obtained by the DLAC solely with M.tb proteins purified without 2D-gel and MS process because the IDs of purified proteins are known. The ligand data obtained by DLAC was applied to improve crystal quality of M.tb proteins to be able to solve their structures 40 .…”

Section: Resultsmentioning

confidence: 99%

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A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

Bhagavat¹,

Kim

et al. 2017

Sci Rep

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Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived from a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.

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“…These aromatic rings promote hydrophobic interactions between Cibacron Blue F3GA and pDNA. The intermolecular electrostatic forces and hydrogen bond formation contribute to the driving force for pDNA adsorption on cryogel discs [29][30][31][32][33].…”

Section: Characterization Of Cryogelmentioning

confidence: 99%