Probing the Interaction between a High-Affinity Single-Chain Fv and a Pyrimidine (6-4) Pyrimidone Photodimer by Site-Directed Mutagenesis†

Biochemistry ◽  
1999 ◽  
Vol 38 (2) ◽  
pp. 532-539 ◽  
Author(s):  
Hiroyuki Kobayashi ◽  
Hiroshi Morioka ◽  
Kunihiro Tobisawa ◽  
Takuya Torizawa ◽  
Koichi Kato ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Single Chain ◽  
High Affinity ◽  
Single Chain Fv

scholarly journals The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuki Kiguchi ◽  
Hiroyuki Oyama ◽  
Izumi Morita ◽  
Yasuhiro Nagata ◽  
Naoko Umezawa ◽  
...  
Keyword(s):  
Affinity Maturation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Affinities ◽  
Diagnostic Systems ◽  
Single Chain ◽  
Single Chain Fv ◽  
Single Chain Fv Fragment ◽  
Framework Region

AbstractIn vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting “hot regions,” i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two “designed” site-directed mutagenesis (A and B) targeted the N-terminal 1–10 positions of the VH framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (Ka, 3.6 × 108 M−1). Mutagenesis A substituted the amino acids at the position 1–3, 5–7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1–6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17–31-fold increased affinity with > 109 M−1Ka values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest Ka value, 1.1 × 1010 M−1.

scholarly journals Mapping the BKCa Channel's “Ca2+ Bowl”

2004 ◽  
Vol 123 (5) ◽  
pp. 475-489 ◽  
Author(s):  
Lin Bao ◽  
Christina Kaldany ◽  
Ericka C. Holmstrand ◽  
Daniel H. Cox
Keyword(s):  
Fusion Protein ◽  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Membrane Domain ◽  
Intracellular Domain ◽  
High Affinity ◽  
Acidic Residues ◽  
The Relationship

There is controversy over whether Ca2+ binds to the BKCa channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca2+ sensitivity act at the point of Ca2+ coordination. One region in the intracellular domain that has been implicated in Ca2+ sensing is the “Ca2+ bowl”. This region contains many acidic residues, and large Ca2+-bowl mutations eliminate Ca2+ sensing through what appears to be one type of high-affinity Ca2+-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca2+ bowl that are most important for Ca2+ sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca2+ binding to a fusion protein that contains a portion of the BKCa channel's intracellular domain. Thus, much of our data supports the conclusion that Ca2+ binds to the BKCa channel's intracellular domain, and they define the Ca2+ bowl's essential Ca2+-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca2+ sensing and those that disrupt Ca2+ binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca2+ bowl may be in a nonnative conformation.

scholarly journals Use of Photoaffinity Labeling and Site-directed Mutagenesis for Identification of the Key Residue Responsible for Extraordinarily High Affinity Binding of UCN-01 in Human α1-Acid Glycoprotein

2004 ◽  
Vol 280 (2) ◽  
pp. 1384-1391 ◽  
Author(s):  
Masaaki Katsuki ◽  
Victor Tuan Giam Chuang ◽  
Koji Nishi ◽  
Kohichi Kawahara ◽  
Hitoshi Nakayama ◽  
...  
Keyword(s):  
Photoaffinity Labeling ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Acid Glycoprotein ◽  
High Affinity

scholarly journals Site-Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor. Arg206 Determines High-Affinity Binding Sites of C5a Receptor

1996 ◽  
Vol 235 (1-2) ◽  
pp. 82-90 ◽  
Author(s):  
Ute Raffetseder ◽  
Detlef Roper ◽  
Laurence Mery ◽  
Claudia Gietz ◽  
Andreas Klos ◽  
...  
Keyword(s):  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Affinity Binding ◽  
C5a Receptor ◽  
High Affinity Binding ◽  
High Affinity ◽  
Charged Residues ◽  
Helical Region

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

Interactions between factor XIII and the αC region of fibrinogen

Blood ◽  
2011 ◽  
Vol 117 (12) ◽  
pp. 3460-3468 ◽  
Author(s):  
Kerrie A. Smith ◽  
Penelope J. Adamson ◽  
Richard J. Pease ◽  
Jane M. Brown ◽  
Anthony J. Balmforth ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Factor Xiii ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enhancing Effect ◽  
High Affinity ◽  
A Subunit ◽  
First Time

Abstract Fibrinogen αC residues 242-424 have been shown to have a major regulatory role in the activation of factor XIII-A2B2 (FXIII-A2B2); however, the interactions underpinning this enhancing effect have not been determined. Here, we have characterized the binding of recombinant (r)FXIII-A subunit and FXIII-A2B2 with fibrin(ogen) and fibrin αC residues 233-425. Using recombinant truncations of the fibrin αC region 233-425 and surface plasmon resonance, we found that activated rFXIII-A bound αC 233-425 (Kd of 2.35 ± 0.09μM) which was further localized to αC 389-403. Site-directed mutagenesis of this region highlighted Glu396 as a key residue for binding of activated rFXIII-A. The interaction was specific for activated rFXIII-A and depended on the calcium-induced conformational change known to occur in rFXIII-A during activation. Furthermore, nonactivated FXIII-A2B2, thrombin-cleaved FXIII-A2B2, and activated FXIII-A2B2 each bound fibrin(ogen) and specifically αC region 371-425 with high affinity (Kd < 35nM and Kd < 31nM, respectively), showing for the first time the potential involvement of the αC region in binding to FXIII-A2B2. These results suggest that in addition to fibrinogen γ′ chain binding, the fibrin αC region also provides a platform for the binding of FXIII-A2B2 and FXIII-A subunit.

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