Evaluation of binding affinity of protein-mutant dna complexes in solution by laser spray mass spectrometry

Abstract

We have applied laser spray mass spectrometry developed by Hiraoka et al. to investigate the binding affinity of protein-mutant DNA complexes. The results were compared with our previous data of collision-induced dissociation (CID) experiments using electrospray ionization mass spectrometry (ESI-MS). Systematic experiments were carried out on the complexes of the c-Myb DNA binding domain (c-Myb DBD) bound to eight kinds of 16-or 22-mer point mutant double-stranded DNA (dsDNA), whose solution K d values are different in the range from 10 Ϫ9 M to 10 Ϫ7 M. The dissociation curve as a function of laser power was plotted for each complex, and the laser power where 50% of complex was dissociated (E 50% ) in population was obtained. The correlation coefficient between E 50% and the relative binding free-energy change (⌬⌬G) of each complex formation in solutions was 0.9808, which is much better than the coefficient obtained by the previous ESI-CID experiments that was 0.859. In addition, complexes of the c-Myb DBD with five other mutant dsDNA were also examined to confirm that laser spray can be used to estimate the K d values of a DNA-protein complex in solutions if an appropriate calibration curve is available. In the process of laser spray, dissociations of these noncovalent complexes occur in solutions, but not in the gas phase. This differs greatly from ESI-CID. Laser spray mass spectrometry has been found to be better than ESI-CID in evaluating binding affinity of a protein to various mutant DNA. (J Am Soc Mass Spectrom 2006, 17, 611-620) © 2006 American Society for Mass Spectrometry E lectrospray ionization mass spectrometry (ESI-MS) has been successfully applied to detect the protonated molecules of noncovalent biomolecular complexes that are involved in various important biological events [1][2][3][4][5][6][7][8]. Although mass spectrometry cannot provide structural information of a biological complex at an atomic level, it can rapidly offer information on the binding stoichiometry, specificity, and stability of the complex with a small amount of the sample. In addition, dissociation of the complex can also be achieved by changing the mass spectrometric parameters. We have previously applied ESI-MS to examine the binding affinity of a protein to various double-stranded DNA (dsDNA) oligomers [9] by using a minimal DNA-binding domain (DBD) of a transcription factor c-Myb [10].The c-Myb DBD, 13 kDa, specifically recognizes dsDNA with the consensus sequence of AACNG (N denotes A or T or G or C) [11][12][13]. We have carried out systematic investigation on the gas-phase stability of the complexes of the c-Myb DBD bound to seven 22-mer dsDNA oligomers. The dsDNA are single-point mutants and the complexes of the c-Myb DBD with dsDNA mutants have different solution K d values in a wide range of 6.3 ϫ 10 Ϫ7 M to 2.8 ϫ 10 Ϫ9 M. Multiply protonated molecules of the complexes were generated by ESI-MS, and successively subjected to collision induced dissociation (CID) in the first vacuum region [9]. A dissociation curve as ...

Denaturation of α‐lactalbumin and ubiquitin studied by electrospray and laser spray

“…This indicates that the laser heating seems less effective than lowering the pH of aqueous solution for the denaturation of this protein under the present experimental conditions. In other words, laser spray enables observation of the intermediate transition states of proteins in an appropriate buffer 17–20…”

“…The general experimental procedures are similar to those described previously 17–20. In brief, a stainless steel capillary (i.d.…”

“…A comparative study for these two compounds was made using electrospray and laser spray. In laser spray developed in our laboratory,17–20 the tip of an electrospray capillary is irradiated with a continuous CO 2 laser beam (10.6 µm). In our previous work, applying a relatively low laser irradiation level (a laser power of ∼2 W and a focal spot size of ∼0.3 mm) resulted in a quiescent and smooth vaporization of aqueous solutions.…”

Biochemical analysis of the interaction between Arabidopsis thaliana AtMYB30 transcription factor and its DNA specific target site

“…Okashi et al (2005) evaluated the binding affinity of complexes between a DNA-binding domain of the transcription factor c-MYB and several double-stranded DNA by electrospray ionization mass spectrometry; K D values in the range of 6.3×10 −7 M to 2.8×10 −9 M, similar to those obtained by us (10 −9 M) were found. Besides, other groups already calculated K D values for MYB proteins using several techniques, ranging from 4.5×10 −9 M (pNgTRF1, a doublestranded telomeric repeat binding factor with a single MYBlike domain of Nicotiana glutinosa, Yang et al 2003); 3.2× 10 −9 M (human TRF1, König et al 1998); 5.5× l0 −7 M using fluorescence anisotropy (Madan et al 1994); 8.0×10 −8 M (confirmed by ITC experiments for Tay1 protein from the yeast Yarrowia lipolytica containing two MYB domains), and 2.8×10 −9 M to 6.3×10 −7 M using mass spectrometry (Shi et al 2006). All these findings confirm that the binding affinity of MYB domains and DNA are clearly around 10 −8 M.…”

[P2–068]: SOLUBLE AMYLOID‐BETA AGGREGATES FROM HUMAN ALZHEIMER's DISEASE BRAINS

“…Many of the previously reported preparations have involved SDS PAGE to assess the size forms, which can both break apart larger Aβ assemblies and cause monomeric Aβ to aggregate ex vivo 42 . A highly cited previous report 23 used size exclusion chromatography to assess the size of the soluble Aβ aggregates, but the running buffer was ammonium acetate pH 8.5 which can cause changes in assembly of protein complexes [43][44][45][46][47] . We found that ammonium acetate pH 8.5 consistently changes the apparent size of soluble Aβ aggregates ( Supplementary Fig.…”