scholarly journals Enhancing ubiquitin crystallization through surface-entropy reduction

2014 ◽  
Vol 70 (10) ◽  
pp. 1434-1442 ◽  
Author(s):  
Patrick J. Loll ◽  
Peining Xu ◽  
John T. Schmidt ◽  
Scott L. Melideo
Keyword(s):  
Surface Density ◽  
Double Mutant ◽  
High Surface ◽  
Wild Type ◽  
Mutant Proteins ◽  
X Ray ◽  
Surface Entropy ◽  
Lysine Residues ◽  
Entropy Reduction ◽  
Packing Interactions

Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

scholarly journals Plasmodium falciparum Kelch13 and its artemisinin-resistant mutants assemble as hexamers in solution: a SAXS data driven shape restoration study

2021 ◽  
Author(s):  
Nainy Goel ◽  
Kanika Dhiman ◽  
Nidhi Kalidas ◽  
Anwesha Mukhopadhyay ◽  
Ashish ◽  
...  
Keyword(s):  
Artemisinin Resistance ◽  
Resistant Mutant ◽  
Data Driven ◽  
Wild Type ◽  
Saxs Data ◽  
Mutant Proteins ◽  
X Ray ◽  
X Ray Scattering ◽  
Shape Restoration ◽  
Spatial Positioning

AbstractArtemisinin-resistant mutations in PfKelch13 identified worldwide are mostly confined to its BTB/POZ and KRP domains. To date, only two crystal structures of the BTB/POZ-KRP domains as tight dimers are available, which limits structure-based interpretations of its functionality. Our solution Small-Angle X-ray Scattering (SAXS) data driven shape restoration of larger length of protein brought forth that: i) PfKelch13 forms a stable hexamer in P6 symmetry, ii) interactions of the N-termini drive the hexameric assembly, and iii) the six KRP domains project independently in space, forming a cauldron-like architecture. While artemisinin-sensitive mutant A578S packed like the wild-type, hexameric assemblies of dominant artemisinin-resistant mutant proteins R539T and C580Y displayed detectable differences in spatial positioning of their BTB/POZ-KRP domains. Lastly, mapping of mutations known to enable artemisinin resistance explained that most mutations exist mainly in these domains because they are non-detrimental to assembly of mutant PfKelch13 and yet can alter the flux of downstream events essential for susceptibility to artemisinin.

Non-destructive X-ray analysis of Arabidopsis embryo mutants

1993 ◽  
Vol 3 (3) ◽  
pp. 167-170 ◽  
Author(s):  
R. J. Bino ◽  
J. W. Aartse ◽  
W. J. van der Burg
Keyword(s):  
Arabidopsis Thaliana ◽  
Double Mutant ◽  
Embryo Morphology ◽  
Wild Type ◽  
X Ray ◽  
Non Destructive ◽  
Mature Seeds

AbstractX-radiography is a simple, rapid and non-destructive method for analysing the morphology of embryos in dry, mature seeds of Arabidopsis thaliana. In wild type seeds, the cotyledons, hypocotyl and radicle tip can be readily distinguished. In seeds of the mutant types knolle, keule, and the double mutant keulelgnom, aberrations in embryo morphology can be visualized. X-radiography may therefore be useful in the isolation of embryo mutants from Arabidopsis seed samples.

scholarly journals The Drosophila melanogaster DmRAD54 Gene Plays a Crucial Role in Double-Strand Break Repair after P-Element Excision and Acts Synergistically with Ku70 in the Repair of X-Ray Damage

1999 ◽  
Vol 19 (9) ◽  
pp. 6269-6275 ◽  
Author(s):  
Rolf Kooistra ◽  
Albert Pastink ◽  
José B. M. Zonneveld ◽  
Paul H. M. Lohman ◽  
Jan C. J. Eeken
Keyword(s):  
Drosophila Melanogaster ◽  
Homologous Recombination ◽  
Crucial Role ◽  
Double Mutant ◽  
Strand Break ◽  
P Element ◽  
Slight Reduction ◽  
Wild Type ◽  
Dsb Repair ◽  
X Ray

ABSTRACT The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogasterstrain deficient in the RAD54 homolog DmRAD54is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54 −/− flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, aDmRad54 −/−/DmKu70 −/−double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements inDrosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3′ overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

scholarly journals The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography

2004 ◽  
Vol 385 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Barbara PETSCHACHER ◽  
Stefan LEITGEB ◽  
Kathryn L. KAVANAGH ◽  
David K. WILSON ◽  
Bernd NIDETZKY
Keyword(s):  
Double Mutant ◽  
Profile Analysis ◽  
Xylose Reductase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binary Complex ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
X Ray ◽  
X Ray Crystallography

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.

scholarly journals Structural studies of a surface-entropy reduction mutant of O-GlcNAcase

2019 ◽  
Vol 75 (1) ◽  
pp. 70-78 ◽  
Author(s):  
Alexandra Males ◽  
Gideon J. Davies
Keyword(s):  
Active Sites ◽  
Crystal Form ◽  
Wild Type ◽  
Post Translational Modification ◽  
Crystal Forms ◽  
Surface Entropy ◽  
Human Enzyme ◽  
C Terminus ◽  
Crystal Contacts ◽  
Entropy Reduction

The enzyme O-GlcNAcase catalyses the removal of the O-GlcNAc co/post-translational modification in multicellular eukaryotes. The enzyme has become of acute interest given the intimate role of O-GlcNAcylation in tau modification and stability; small-molecular inhibitors of human O-GlcNAcase are under clinical assessment for the treatment of tauopathies. Given the importance of structure-based and mechanism-based inhibitor design for O-GlcNAcase, it was sought to test whether different crystal forms of the human enzyme could be achieved by surface mutagenesis. Guided by surface-entropy reduction, a Glu602Ala/Glu605Ala variant [on the Gly11–Gln396/Lys535–Tyr715 construct; Roth et al. (2017), Nature Chem. Biol. 13, 610–612] was obtained which led to a new crystal form of the human enzyme. An increase in crystal contacts stabilized disordered regions of the protein, enabling 88% of the structure to be modelled; only 83% was possible for the wild-type construct. Although the binding of the C-terminus was consistent with the wild type, Lys713 in monomer A was bound in the −1 subsite of the symmetry-related monomer A and the active sites of the B monomers were vacant. The new crystal form presents an opportunity for enhanced soaking experiments that are essential to understanding the binding mechanism and substrate specificity of O-GlcNAcase.

scholarly journals Separate Ion Pathways in a Cl−/H+ Exchanger

2005 ◽  
Vol 126 (6) ◽  
pp. 563-570 ◽  
Author(s):  
Alessio Accardi ◽  
Michael Walden ◽  
Wang Nguitragool ◽  
Hariharan Jayaram ◽  
Carole Williams ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Transport Mechanism ◽  
Double Mutant ◽  
Wild Type ◽  
Extracellular Solution ◽  
X Ray ◽  
Single Mutant ◽  
Cl Transport ◽  
Alternating Access

CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl−. A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

2007 ◽  
Vol 292 (3) ◽  
pp. G818-G828 ◽  
Author(s):  
Kaori Mochizuki ◽  
Tatehiro Kagawa ◽  
Asano Numari ◽  
Matthew J. Harris ◽  
Johbu Itoh ◽  
...  
Keyword(s):  
Protein Stability ◽  
Bile Salt ◽  
Intracellular Trafficking ◽  
Double Mutant ◽  
Yellow Fluorescent Protein ◽  
Bile Salt Export Pump ◽  
Transport Activity ◽  
Wild Type ◽  
Mutant Proteins ◽  
Quadruple Mutant

The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn109, Asn116, Asn122, and Asn125) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A1 extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.

scholarly journals Conformation of the AcrB Multidrug Efflux Pump in Mutants of the Putative Proton Relay Pathway

2006 ◽  
Vol 188 (20) ◽  
pp. 7290-7296 ◽  
Author(s):  
Chih-Chia Su ◽  
Ming Li ◽  
Ruoyu Gu ◽  
Yumiko Takatsuka ◽  
Gerry McDermott ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Mutant Proteins ◽  
X Ray ◽  
Proton Relay ◽  
Initial Binding

ABSTRACT We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.

scholarly journals The Role of Surface Exposed Lysine in Conformational Stability and Functional Properties of Lipase from Staphylococcus Family

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3858
Author(s):  
Nurul Nadirah Ahmad ◽  
Nor Hafizah Ahmad Kamarudin ◽  
Adam Thean Chor Leow ◽  
Raja Noor Zaliha Raja Abd. Rahman
Keyword(s):  
Double Mutant ◽  
Enzyme Stability ◽  
Conformational Stability ◽  
Conformational Dynamics ◽  
Water Environment ◽  
Relative Activity ◽  
Wild Type ◽  
Single Mutant ◽  
Lysine Residues ◽  
The Stability

Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 °C. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 °C change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 °C (T1/2 = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.

scholarly journals Structural and kinetic analyses of the H121A mutant of cholesterol oxidase

2006 ◽  
Vol 400 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Louis Lim ◽  
Gianluca Molla ◽  
Nicole Guinn ◽  
Sandro Ghisla ◽  
Loredano Pollegioni ◽  
...  
Keyword(s):  
Redox Potential ◽  
Cholesterol Oxidase ◽  
Isomerization Reaction ◽  
Planar Geometry ◽  
Tryptophan Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Turnover Number ◽  
Mutant Proteins ◽  
X Ray

Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a ≈100 mV decrease in the midpoint redox potential and a ≈40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024–18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 Å resolution (1 Å≡0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180° about χ1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.

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