Crystallization and Preliminary X-ray Analysis of a Mutant Duck δ II Crystallin

Abu-Abed, Mona; Turner, Mary Ann; Atkinson, Jennifer; Dole, Kiran; Howell, P. Lynne

doi:10.1006/jmbi.1994.1694

Cited by 5 publications

(3 citation statements)

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“…Both the closely associated monomers and the tetramer are held together by mainly hydrophobic interactions along the length of helices h8, h11, h12. The overall structure of the tetramer exhibits 222 noncrystallographic symmetry as previously observed in the self-rotation function (Abu-Abed et al, 1994).…”

Section: Resultssupporting

confidence: 61%

“…Crystallization and Data Collection. The initial crystallization conditions found for duck δ-crystallin have been described elsewhere (Abu-Abed et al, 1994). However, prior to data collection at the Cornell High Energy Synchrotron 1) was collected at room temperature on a Siemen's Xentronics area detector with a Rigaku RU200 rotating anode X-ray generator (λ ) 1.541 Å) and processed using the XDS software package (Blum et al, 1987;Kabsch, 1988a,b).…”

Section: Gene Expression Purification Crystallization and Datamentioning

confidence: 99%

“…Examination of the available protein sequences for δ I and δ II crystallins reveals an 88−94% identity among the various δ-crystallins and between 65% and 71% identity to the metabolic enzyme, argininosuccinate lyase (ASL) (EC 4.3.2.1) (Mori et al, 1990; O'Brien et al, 1986; Piatigorsky et al, 1988). Since both δ-crystallins are preferentially expressed in the lens tissue and as they are more identical to each other than to human ASL, it appears that the recruitment of ASL to an eye lens role must have preceded the gene duplication event, demonstrating that gene duplication is not a mandatory prerequisite for the evolution of a new protein function (Hughes, 1994; Kimura & Ohta, 1974; Piatigorsky & Wistow, 1989, 1991).…”

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Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91^,

Abu-Abed

Turner²,

Vallée³

et al. 1997

Biochemistry

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The major soluble protein component of avian and reptilian eye lenses, delta crystallin, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses there are two highly homologous delta crystallins, termed delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I crystallin is inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H91N) has been determined to 2.5 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft between three different monomers of the tetrameric protein. A reexamination of the kinetic properties of the H91N mutant reveals that the mutant has 10% wild-type activity. The Vmax of the mutant protein is identical to that of the wild-type protein, but a 10-fold increase in the Michaelis constant is seen, suggesting that His 91 is involved in binding the substrate. In an effort to determine the reasons for the loss of enzymatic activity in delta I crystallin, a structural comparison of the H91N mutant with the enzymatically inactive turkey delta I crystallin has been performed. This study revealed a remarkable similarity in the overall structures of the two proteins. Three regions of secondary structure do differ significantly between the two models; these include the N-terminal tail, a loop containing residues 76-91, and a cis versus trans peptide linkage at residue Thr 322. The cis to trans peptide variation appears to be an interspecies difference between turkey and duck and is therefore not directly involved in the loss of enzymatic activity. All the residues implicated in the catalytic mechanism are conserved in both the active and inactive proteins, and given the linearity of the relationship between the enzymatic activity of duck delta I/delta II heterotetramers and their delta II content (Piatigorsky & Horwitz, 1996), it is evident from the structure that only one of the three domains that contributes to the active site is responsible for the loss of activity in the delta I protein. Given the structural differences found in domain 1 (N-terminal tail and 76-91 loop), we postulate that these differences are responsible for the loss of catalytic activity in the delta I crystallin protein and that the delta I protein is inactive because it no longer binds the substrate.

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Section: Resultssupporting

confidence: 61%

Section: Gene Expression Purification Crystallization and Datamentioning

confidence: 99%

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confidence: 99%

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Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91^,

Abu-Abed

Turner²,

Vallée³

et al. 1997

Biochemistry

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Influence of divalent cations in protein crystallization

Trakhanov

Quiocho

1995

Protein Sci.

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We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/ isoleucine/valine-binding protein (LIVBP) (M, = 36,700) and leucine-specific binding protein (LBP) (M, = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells. Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality. This comes about 15 years after the ligand-free proteins were crystallized. Nine other different divalent cations were tried as additives in the crystallization of LIVBP with polyethylene glycol 8000 as precipitant, and each showed different effects on the crystal quality and morphology. Cd2+ produced large hexagonal prism crystals of LIVBP, whereas a majority of the cations resulted in less desirable needle-shaped crystals. Zn2+ gave crystals that are long rods with hexagonal cross sections, a shape intermediate between the hexagonal prism and needle forms. The concentration of Cd2+ is critical. The best crystals of the LIVBP were obtained in the presence of 1 mM CdCI2, whereas those of LBP, with trigonal prism morphology, were obtained at a much higher concentration of 100 mM. Both crystals diffract to at least 1.7 A resolution using a conventional X-ray source.Keywords: active transport receptors; cation effects; protein crystallization; X-ray crystallography Since the crystals of ligand-free leucine/isoleucine/valinebinding protein were obtained 17 years ago (Meador & Quiocho, 1978) and leucine-specific-binding protein 4 years later (Trakhanov et al., 1982), we have attempted on several occasions to crystallize these proteins in the presence of ligands. (The crystal structures of the ligand-free proteins have since been determined [Sack et al., 1989a.) These attempts have, until recently, met with failure. Other investigators have earlier reported obtaining crystals of LIVBP in the presence of leucine. For example, crystallization from ammonium sulfate solution used as a final step in the LIVBP purification resulted in showers of very thin needle-shaped crystals (Anraku, 1968;Penrose et al., 1968). Oxender and Quay (1975) have also obtained crystals from 2-methyl-2,4-pentanediol solution in the form of small (around 50 pm size) hexagonal prisms. We have reproduced these crystal forms and have also been able to grow very thin rod crystals of LIVBP in the presence of leucine using organic precipitants such as ethanol or isopropanol (unpubl. results). All these initial crystals were, however, unsuitable for crystallographic analysis. Moreover, our many trials to improve the qual- Abbreviations: LIVBP, leucine/isoleucine/valine-binding protein; LBP, leucine-specific binding protein; HBP, histidine-binding protein; MPD, 2-methyl-2,4-pentanediol; PEG, polyethylene glycol. ity of these crystal forms led to nowhere. Conditions that were varied include type and concentration of precipitating agents, protein concentration, type (leucin...

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pH-Induced Reversible Dissociation of Tetrameric Duck Lens Delta-Crystallin

Chang

Lee

Chow

1997

Experimental Eye Research

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Crystallization and Preliminary X-ray Analysis of a Mutant Duck δ II Crystallin

Cited by 5 publications

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Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91^,

Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91^,

Influence of divalent cations in protein crystallization

pH-Induced Reversible Dissociation of Tetrameric Duck Lens Delta-Crystallin

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Crystallization and Preliminary X-ray Analysis of a Mutant Duck δ II Crystallin

Cited by 5 publications

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Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91,

Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91,

Influence of divalent cations in protein crystallization

pH-Induced Reversible Dissociation of Tetrameric Duck Lens Delta-Crystallin

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Product

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Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91^,

Structural Comparison of the Enzymatically Active and Inactive Forms of δ Crystallin and the Role of Histidine 91^,