Protein purity and yield are two critical parameters for successful protein characterization using structural techniques such as X-ray crystallography, NMR, and several other biophysical methods. The ...yeast Saccharomyces cerevisiae is one of the popular eukaryotic model systems for overexpression and subsequent purification of recombinant proteins. Here, we describe a protocol for cloning, overexpression, purification, and crystallization of arrestin-1 and its splice variant p44 from yeast. The purification protocol involves a single-affinity chromatography step on a Strep-Tactin column. Highly purified arrestins can be concentrated up to 15mg/mL using ultrafiltration and can be stored in the frozen state for several months without any loss of functionality.
Unique features of Light-Oxygen-Voltage (LOV) proteins like relatively small size (~12-19 kDa), inherent modularity, highly-tunable photocycle and oxygen-independent fluorescence have lately been ...exploited for the generation of optical tools. Structures of LOV domains reported so far contain a flavin chromophore per protein molecule. Here we report two new findings on the short LOV protein W619_1-LOV from Pseudomonas putida. First, the apo-state crystal structure of W619_1-LOV at 2.5 Å resolution reveals conformational rearrangements in the secondary structure elements lining the chromophore pocket including elongation of the Fα helix, shortening of the Eα-Fα loop and partial unfolding of the Eα helix. Second, the apo W619_1-LOV protein binds both natural and structurally modified flavin chromophores. Remarkably different photophysical and photochemical properties of W619_1-LOV bound to 7-methyl-8-chloro-riboflavin (8-Cl-RF) and lumichrome imply application of these variants as novel optical tools as they offer advantages such as no adduct state formation, and a broader choice of wavelengths for in vitro studies.
Malaria causes millions of death cases per year. Since Plasmodium falciparum rapidly develops drug resistance, it is of high importance to investigate potential drug targets which may lead to novel ...rational therapy approaches. Here we report on the interaction of translationally controlled tumor protein of P. falciparum (PfTCTP) with the anti-malarial drug artemisinin. Furthermore, we investigated the crystal structure of PfTCTP. Using mass spectrometry, bioinformatic approaches and surface plasmon resonance spectroscopy, we identified novel binding sites of artemisinin which are in direct neighborhood to amino acids 19–46, 108–134 and 140–163. The regions covered by these residues are known to be functionally important for TCTP function. We conclude that interaction of artemisinin with TCTP may be at least in part explain the antimalarial activity of artemisinin.
The human membrane‐bound α/β‐hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's ...diseases, through the degradation of the key lipid messenger 2‐arachidonylglycerol (2‐AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2‐AG‐hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane‐bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2‐AG and esters of medium‐length fatty acids. PA2949 in the presence of n‐octyl β‐d‐glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6‐like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult‐to‐crystallize proteins.
Homologous expression of the membrane‐bound esterase PA2949 from Pseudomonas aeruginosa PA01 and the purification of detergent‐solubilized enzyme resulted in stable PA2949 protein that crystallized. The crystals obtained were used for X‐ray analysis and diffracted to a resolution of 2.74 Å.
Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota. This family of ...photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 Å resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.
Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present ...the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 Å reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P-3 is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.
The peptide amidase from
Stenotrophomonas maltophilia catalyses predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Peptide bonds or amide functions in amino acid side-chains ...are not hydrolysed. This specificity makes peptide amidase (Pam) interesting for different biotechnological applications. Pam belongs to the amidase signature (AS) family. It is the first protein within this family whose tertiary structure has been solved. The structure of the native Pam has been determined with a resolution of 1.4
Å and in complex with the competitive inhibitor chymostatin at a resolution of 1.8
Å. Chymostatin, which forms acyl adducts with many serine proteases, binds non-covalently to this enzyme.
Pam folds as a very compact single-domain protein. The AS sequence represents a core domain that is covered by α-helices. This AS domain contains the catalytic residues. It is topologically homologous to the phosphoinositol phosphatase domain.
The structural data do not support the recently proposed Ser-Lys catalytic dyad mechanism for AS enzymes. Our results are in agreement with the role of Ser226 as the primary nucleophile but differ concerning the roles of Ser202 and Lys123: Ser202, with direct contact both to the substrate molecule and to Ser226, presumably serves as an acid/bases catalyst. Lys123, with direct contact to Ser202 but no contact to Ser226 or the substrate molecule, most likely acts as an acid catalyst.
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