Site-specific mutagenesis of human apolipoprotein E. Receptor binding activity of variants with single amino acid substitutions.

AbstractCdk1 has been found to phosphorylate the majority of its substrates in disordered regions. These phosphorylation sites do not appear to require precise positioning for their function. The mitotic kinesin-5 Cin8 was shown to be phosphoregulated at three Cdk1 sites in disordered loops within its catalytic motor domain. Here, we examined the flexibility of Cin8 phosphoregulation by analyzing the phenotypes of synthetic Cdk1-sites that were systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites that we created, eight were non-functional; 19 were neutral, similar to the phosphodeficient variant; and two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one site resulted in novel phosphoregulation, and only one site, immediately adjacent to a native Cdk1 site, produced phosphoregulation similar to wild-type. This study shows that, while the gain of a single phosphorylation site can confer regulation and modulate the dynamics of the spindle, to achieve optimal regulation of a mitotic kinesin-5 motor protein, phosphoregulation has to be site-specific and precise.

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INTERFERON SEQUENCE HOMOLOGY AND RECEPTOR BINDING ACTIVITY OF OVINE TROPHOBLAST ANTILUTEOLYTIC PROTEIN

Journal of Endocrinology ◽

10.1677/joe.0.115r013 ◽

1987 ◽

Vol 115 (2) ◽

pp. R13-R15 ◽

Cited By ~ 108

Author(s):

H.J. Stewart ◽

S.H.E. McCann ◽

P.J. Barker ◽

K.E. Lee ◽

G.E. Lamming ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Receptor Binding ◽

Early Pregnancy ◽

Sequence Homology ◽

Binding Activity ◽

Membrane Receptors ◽

Interferon Α ◽

Receptor Binding Activity ◽

Terminal Amino

ABSTRACT Sequencing of the 40 N-terminal amino acids of the blastocyst protein responsible for blocking corpus luteum regression in early pregnancy in sheep revealed a 37% homology with human α-interferon; 28% of the remaining amino acid changes were conservative. 125I-Labelled human α-interferon bound to membrane receptors from sheep uteri with an approximate Kd of 4 × 10−11 M; binding was inhibited by unlabelled α-interferon or purified blastocyst antiluteolytic protein. The blastocyst antiluteolytic protein therefore closely resembles the interferon-α family of antiviral proteins.

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A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2006.03.139 ◽

2006 ◽

Vol 344 (1) ◽

pp. 106-113 ◽

Cited By ~ 13

Author(s):

Yuxian He ◽

Jingjing Li ◽

Shibo Jiang

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Amino Acid Substitution ◽

Binding Activity ◽

Single Amino Acid Substitution ◽

Spike Protein ◽

Single Amino Acid ◽

Antigenic Structure ◽

Receptor Binding Domain ◽

Sars Coronavirus

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Phenotypic Analysis of Random hnsMutations Differentiate DNA-Binding Activity from Properties offimA Promoter Inversion Modulation and Bacterial Motility

Journal of Bacteriology ◽

10.1128/jb.181.3.941-948.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 941-948 ◽

Cited By ~ 18

Author(s):

Gina M. Donato ◽

Thomas H. Kawula

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Constitutive Expression ◽

Binding Activity ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Phenotypic Analysis ◽

Mutator Strain ◽

Dna Binding Activity ◽

And Function

ABSTRACT H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of theproU and fimB genes, increased fimApromoter inversion rates, and repression of the flhCDmaster operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proUexpression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimBoccurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hnsmutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.

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