Herrmann, Ch.

Kerstan, A.

Ladnorg, T.

Vöpel, T.

Wöll, Ch.

Zacher, D.

KITopen

KIT – Universität des Landes Baden-Württemberg undnationales Forschungszentrum in der Helmholtz-GemeinschaftHGBP1 as a model system investigated by several surface techniquesInstitute of Functional InterfacesChemistry of oxydic and organic InterfacesA. Kerstan1, T.Ladnorg2, T. Vöpel1, D.Zacher3, Ch.Herrmann1, Ch.Wöll2in solution at the surfaceMonomerDimer• Solution: Dimer activity• Surface: two possibilities for   reduced activity Diffusion limitiation Monomer activity• Exception: Q577C/K485C Dimerisation at the surfaceTests of activity on the surface with SPRAnalysis of thickness with the AFMHigh correlation• Orientation:Single mutant: perpendicular tosurfaceDouble mutant: parallel to surface• Dimerisation:(-) Single mutant: Q577C(+) Single mutant: K485C(+) Double mutant: Q577C/K485C4 µm 4 µm 4 µmStreptavidin K485C wtAFM on lateral structured surfacesIntroductionIn medical technology concerning the surface immobilizationof proteins in a defined orientation, maintaining their activityis a critical aspect. This protein, hGBP1 bearing GTPasecatalytic activity, is furnished either with one biotin anchor atone end of the molecule or with two biotin anchors opposingeach other at the long ends. Various complementarymethods (SPR, AFM and QCM) yield the same conclusion:Immobilisation of hGBP1-mutants• Single mutants go on surface in comparison to the double mutants at themolar ratio of 3:1 • Double and Single mutants have different             FootprintsSingle mutants: approx. 12 nm²Double mutants: approx. 39 nm²Studies of kinetiks of dimerisationStudies of kinetiks of dimerisation of hGBP1• linear regression: - calculation of konout of  slope - koff/kon = KDKD = 7,45 µM (koff out of y-intercept: 6,32 µM)• Plateau value (Monomer-Dimer-equilibrium):  KD = 8,89 µMLineare regressionPlateau valueA. Kerstan, T. Ladnorg, C. Grunwald, T. Vopel, D. Zacher, C. Herrmann, C. Wöll, Biointerphases 2010, 5, 131.1 Department of Physical Chemistry I, University of Bochum, 44801 Bochum, Germany2 Institute of Functional Interfaces, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany3 Department of Anorganic Chemistry II, University of Bochum, 44801 Bochum, Germany K485C : hGBP1 wt  1 : 0,5Q577C/K485C :  hGBP1 wt  1 : 0,8Q577C : hGBP1 wt no dimerisation Cysteinspecific BiotinylationDimerisation of hGBP1 Comparison of hGBP1 mutants in SPR(1) the double-anchored hGBP1 is oriented alongside and (2)the single-anchored protein is oriented upright. In addition,the catalytic activity of hGBP1 reveals full integrity of thesurface bound protein and it also shows that the maximumactivity of hGBP1 is not reached because immobilizationprevents hGBP1 from homo dimer formation in the requiredorientation.Crystal structure of hGBP1 in ribbonpresentation with different anchorpositionsSolutionSurfacetime [min]time [min]time [min] time [min]crystal structure of hGBP1 in ribbon presentationtime [min] time [min]

HGBP1 as a model system investigated by several surface techniques

https://publikationen.bibliothek.kit.edu/220085097/3815917

HGBP1 as a model system investigated by several surface techniques

Abstract

Similar works

Full text

Available Versions

KITopen