PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 16511228

Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation.

Bernard Collins·Raymond C Stevens·Rebecca Page
View original source ↗Export metadata ↓

Abstract

An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ;optimum-solubility' buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 A) structure determination.

Explore related subjects

Archaeoglobus fulgidusBuffersCrystallizationCrystallography, X-RayGenomicsHydrogen-Ion ConcentrationProtein ConformationProteinsSolubilityTemperatureJournal ArticleResearch Support, N.I.H., Extramural

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Bernard Collins, Raymond C Stevens, Rebecca Page. 2005-11-05. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation.. https://doi.org/10.1107/s1744309105035244

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

The role of specific 2'-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA.

The role of 2'-hydroxyl groups in stabilizing the tightly kinked geometry of the kink-turn (K-turn) has been investigated. Individual 2'-OH groups have been removed by chemical synthesis, and the kinking of the RNA has been studied by gel electrophoresis and fluorescence resonance energy transfer. The results have been analyzed by reference to a database of 11 different crystallographic structures of K-turns. The potential hydrogen bonds fall into several classes. The most important are those in the core of the turn and ribose-phosphate interactions around the bulge. Of these the single most important hydrogen bond is one donated from the 2'-OH of the 5' nucleotide of the bulge to the N1 of the adenine of the kink-proximal A*G pair. This is present in all known K-turn structures, and removal of the 2'-OH completely prevents metal ion-induced folding. Hydrogen bonds formed in the minor grooves of the helical stems are less important, and removal of the participating 2'-OH groups leads to reduced impairment of folding. These interactions are generally more polymorphic, and hydrogen bonds probably form where possible, as permitted by the global structure.

Archaeoglobus fulgidus↗
PubMed2006

Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases.

Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 A using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes.

Archaeoglobus fulgidus↗
PubMed2006

Superoxide reduction by Archaeoglobus fulgidus desulfoferrodoxin: comparison with neelaredoxin.

Superoxide reductases (SORs) are non-heme iron-containing enzymes that remove superoxide by reducing it to hydrogen peroxide. The active center of SORs consists of a ferrous ion coordinated by four histidines and one cysteine in a square-pyramidal geometry. In the 2Fe-SOR, a distinct family of SORs, there is an additional desulforedoxin-like site that does not appear to be involved in SOR activity. Our previous studies on recombinant Archaeoglobus fulgidus neelaredoxin (1Fe-SOR) have shown that the reaction with superoxide involves the formation of a transient ferric form that, upon protonation, decays to yield an Fe(3+)-OH species, followed by binding of glutamate to the ferric ion via replacement of hydroxide (Rodrigues et al. in Biochemistry 45:9266-9278, 2006). Here, we report the characterization of recombinant desulfoferrodoxin from the same organism, which is a member of the 2Fe-SOR family, and show that the steps involved in the superoxide reduction are similar in both families of SOR. The electron donation to the SOR from its redox partner, rubredoxin, is also presented here.

Archaeoglobus fulgidus↗