Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C

Kozak, Maciej; Jankowska, Elżbieta; Janowski, Robert; Grzonka, Zbigniew; Grubb, Anders; Fernandez, Marcia Alvarez; Abrahamson, Magnus; Jaskólski, Mariusz

doi:10.1107/s090744499901121x

Cited by 29 publications

(26 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…24 Crystallization of the N-truncated protein was described for the first time by Kozak et al 23 Later, a new polymorphic form was obtained using the same crystallization conditions (0.4 M (NH 4 )H 2 PO 4 , protein concentration 6 mg/ml, 198 C). The new crystals belong to the orthorhombic system, space group C222 1 , with unit cell parameters a ¼ 97.2, b ¼ 99.6, c ¼ 206.1 Å .…”

Section: Methodsmentioning

confidence: 99%

Domain Swapping in N-truncated Human Cystatin C

Janowski

Abrahamson

Grubb

et al. 2004

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Domain Swapping in N-truncated Human Cystatin C

Janowski

Abrahamson

Grubb

et al. 2004

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

“…Cells were cultured at 30°C in a supplemented phosphate-buffered minimal medium ("Kozak" medium), consisting of 87 mM Na 2 HPO 4 , 46 mM KH 2 PO 4 , 18 mM NaCl, 7.5 mM (NH 4 ) 2 SO 4 , 0.2% glucose, 1.7 mM MgSO 4 , 0.117 mM CaCl 2 , 0.015 mM FeSO 4 , and 0.075 mM thiamine (26). In some experiments cells were grown on modified G56 minimal medium, which contains 45 mM HEPES, pH 7.4, 0.3 mM KH 2 PO 4 , 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.2% glucose, 1.7 mM MgSO 4 , 0.117 mM CaCl 2 , 0.015 mM FeSO 4 , and 0.075 mM thiamine (27).…”

Section: Materials-mentioning

confidence: 99%

A Triple Mutant of Escherichia coli Lacking Secondary Acyl Chains on Lipid A

Vorachek-Warren¹,

Ramirez²,

Cotter³

et al. 2002

Journal of Biological Chemistry

121

165

View full text Add to dashboard Cite

All possible combinations of insertion mutations in the three genes encoding the acyl carrier protein-dependent late acyltransferases of lipid A biosynthesis, designated lpxL(htrB), lpxM(msbB), and lpxP, were generated in Escherichia coli K12 W3110. Mutants defective in lpxM synthesize penta-acylated lipid A molecules and grow normally. Strains lacking lpxP fail to incorporate palmitoleate into their lipid A at 12°C but make normal amounts of hexa-acylated lipid A and are viable. Although lpxL mutants and lpxL lpxM double mutants grow slowly on minimal medium at all temperatures, they do not grow on nutrient broth above 32°C. Such mutants retain the ability to synthesize some penta-and hexa-acylated lipid A molecules because of limited induction of lpxP at 30°C but not above 32°C. MKV15, an E. coli lpxL lpxM lpxP triple mutant, likewise grows slowly on minimal medium at all temperatures but not on nutrient broth at any temperature. MKV15 synthesizes a lipid A molecule containing only the four primary (R)-3-hydroxymyristoyl chains. The outer membrane localization and content of lipid A are nearly normal in MKV15, as is the glycerophospholipid and membrane protein composition. However, the rate at which the tetra-acylated lipid A of MKV15 is exported to the outer membrane is reduced compared with wild type. The integrity of the outer membrane of MKV15 is compromised, as judged by antibiotic hypersensitivity, and MKV15 undergoes lysis following centrifugation. MKV15 may prove useful as a host strain for expressing late acyltransferase genes from other Gram-negative bacteria, facilitating the re-engineering of lipid A structure in living cells and the design of novel vaccines.

show abstract

“…Since then, 3D domain swapping has been demonstrated in two other amyloidogenic proteins, namely the prion protein [27] and β‐microglobulin [28]. The interest in HCC, however, has not declined, partly because the dimeric protein has been crystallized in several forms [29], including a polymorph in which the domain‐swapped molecules have aggregated to build an infinite structure with all β‐chains in a perpendicular orientation relative to a common direction [21], as required by the cross‐β canon [30] of amyloid fibril architecture. Although the present view of amyloid aggregation is more complex [31], the interest in 3D domain swapping remains high.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of human cystatin C stabilized against amyloid formation

et al. 2010

Self Cite

View full text Add to dashboard Cite

Human cystatin C (HCC) is a family 2 cystatin inhibitor of papain‐like (C1) and legumain‐related (C13) cysteine proteases. In pathophysiological processes, the nature of which is not understood, HCC is codeposited in the amyloid plaques of Alzheimer’s disease or Down’s syndrome. The amyloidogenic properties of HCC are greatly increased in a naturally occurring L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult life. In all crystal structures of cystatin C studied to date, the protein has been found to form 3D domain‐swapped dimers, created through a conformational change of a β‐hairpin loop, L1, from the papain‐binding epitope. We have created monomer‐stabilized human cystatin C, with an engineered disulfide bond (L47C)–(G69C) between the structural elements that become separated upon domain swapping. The mutant has drastically reduced dimerization and fibril formation properties, but its inhibition of papain is unaltered. The structure confirms the success of the protein engineering experiment to abolish 3D domain swapping and, in consequence, amyloid fibril formation. It illustrates for the first time the fold of monomeric cystatin C and allows verification of earlier predictions based on the domain‐swapped forms and on the structure of chicken cystatin. Importantly, the structure defines the so‐far unknown conformation of loop L1, which is essential for the inhibition of papain‐like cysteine proteases.

show abstract

Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C

Cited by 29 publications

References 24 publications

Domain Swapping in N-truncated Human Cystatin C

Domain Swapping in N-truncated Human Cystatin C

A Triple Mutant of Escherichia coli Lacking Secondary Acyl Chains on Lipid A

Crystal structure of human cystatin C stabilized against amyloid formation

Contact Info

Product

Resources

About