Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of approximately 60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.

A genetically modified form of the human DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.

We have investigated the role of tyrosine phosphorylation of the cyclin-dependent kinase (cdk) inhibitor p27 Kip1 using the acute promyelocytic leukemia cell line NB4 together with granulocyte colonystimulating factor (G-CSF). Short-term G-CSF stimulation resulted in a rapid tyrosine dephosphorylation of p27 Kip1 accompanied by a change in its binding preferences to cdks. On G-CSF stimulation, p27 Kip1 dissociated from cdk4 and associated with cdk2. Binding assays with recombinant p27 Kip1 confirmed that tyrosine-phosphorylated p27 Kip1 preferentially bound to cdk4, whereas unphosphorylated protein preferentially associated with cdk2. In addition, studies with p27 Kip1 point mutations revealed a decisive role of Tyr88 and Tyr89 in binding to cdk4. Furthermore, phosphorylation of Tyr88 and Tyr89 was accompanied by strong nuclear translocation of p27 Kip1 .Taken together, this report provides the first evidence that tyrosine phosphorylation of p27 Kip1 plays a crucial role in binding to cdks and its subcellular localization. Moreover, both effects are mediated by application of G-CSF. (Blood.