From b27a8a4ae2781401889850f114fe2491b81b3476 Mon Sep 17 00:00:00 2001 From: Alexander Ralph Michael Minges Date: Mon, 6 Sep 2010 15:09:16 +0200 Subject: [PATCH] Ergebnisse: CD-Spektroskopie --- .directory | 5 + Bibliothek.bib | 13 + bachelorarbeit.kilepr | 52 +- bachelorarbeit.pdf | Bin 7092038 -> 7148824 bytes bachelorarbeit.tex | 10 +- img/ergebnisse/cd_spektroskopie/spektrum.pdf | Bin 0 -> 56103 bytes img/ergebnisse/cd_spektroskopie/spektrum.svg | 1119 ++++++++++++++++++ inc/abkuerzungen.tex | 2 +- inc/ergebnisse.tex | 16 +- inc/material.tex | 5 +- 10 files changed, 1191 insertions(+), 31 deletions(-) create mode 100644 .directory create mode 100644 img/ergebnisse/cd_spektroskopie/spektrum.pdf create mode 100644 img/ergebnisse/cd_spektroskopie/spektrum.svg diff --git a/.directory b/.directory new file mode 100644 index 0000000..18a5441 --- /dev/null +++ b/.directory @@ -0,0 +1,5 @@ +[Dolphin] +Timestamp=2010,9,5,22,55,8 + +[Settings] +ShowDotFiles=true diff --git a/Bibliothek.bib b/Bibliothek.bib index 0a80b86..c7ede98 100644 --- a/Bibliothek.bib +++ b/Bibliothek.bib @@ -336,6 +336,19 @@ year = {2007} }, +@article{provencher_estimation_1981, + title = {Estimation of globular protein secondary structure from circular dichroism}, + volume = {20}, + issn = {0006-2960}, + url = {http://pubs.acs.org/doi/abs/10.1021/bi00504a006}, + doi = {10.1021/bi00504a006}, + number = {1}, + journal = {Biochemistry}, + author = {Stephen W. Provencher and Juergen Gloeckner}, + year = {1981}, + pages = {33--37} +}, + @book{riedel_anorganische_2007, address = {Berlin; New York}, edition = {7. Aufl.}, diff --git a/bachelorarbeit.kilepr b/bachelorarbeit.kilepr index f56bd7c..4bf7347 100644 --- a/bachelorarbeit.kilepr +++ b/bachelorarbeit.kilepr @@ -4,7 +4,7 @@ img_extIsRegExp=false img_extensions=.eps .jpg .jpeg .png .pdf .ps .fig .gif kileprversion=2 kileversion=2.0.85 -lastDocument=inc/material.tex +lastDocument=inc/ergebnisse.tex masterDocument= name=Bachelorarbeit pkg_extIsRegExp=false @@ -74,10 +74,10 @@ ReadWrite=true [item:Bibliothek.bib] archive=true -column=58 +column=35 encoding=UTF-8 highlight=BibTeX -line=4 +line=338 mode=BibTeX open=true order=5 @@ -94,10 +94,10 @@ order=-1 [item:bachelorarbeit.tex] archive=true -column=0 +column=19 encoding=UTF-8 highlight=LaTeX -line=19 +line=5 mode=LaTeX open=true order=0 @@ -142,6 +142,16 @@ mode= open=false order=-1 +[item:img/ergebnisse/cd_spektroskopie/spektrum.pdf] +archive=true +column=0 +encoding= +highlight= +line=0 +mode= +open=false +order=-1 + [item:img/ergebnisse/expression_reinigung/blot_17_08_10_beschriftet.pdf] 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%Dokumentenklasse +\documentclass[a4paper,fontsize=13pt,utf8x,titlepage=true,toc=bibliography,captions=tableheading,DIV=12,BCOR=1cm]{scrreprt} %Dokumentenklasse \pdfcompresslevel=9 -\usepackage[utf8]{inputenc} % Eingabekodierung UTF-8 +\usepackage[utf8x]{inputenc} % Eingabekodierung UTF-8 \usepackage[T1]{fontenc} % Schriftkodierung -\usepackage{tipa} % Phonetische Symbole (z.B. %textbeta) +%\usepackage{tipa} % Phonetische Symbole (z.B. %textbeta) \usepackage{libertine} % Schriftarten 'Linux Libertine' und 'Linux Biolinum' -\usepackage[ngerman]{babel} % Neue Deutsche Rechtschreibung/Silbentrennung +\usepackage[ngerman]{babel} % Neue Deutsche Rechtschreibung/Silbentrennung mit griechischen Buchstaben \usepackage{setspace} % Paket für das Setzen des Zeilenabstandes \onehalfspacing \usepackage{acronym} % Abkürzungen und Abkürzungsverzeichnis @@ -17,7 +17,7 @@ \newunit{\psi}{psi} % Definition eigener Einheiten: Pounds per inch \newunit{\CV}{CV} -\usepackage{amsmath,amsfonts} 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+105,26 @@ Nachdem sich im photometrischen Bindungsassay der kleinste K$_\text{d}$-Wert bei \includegraphics[width=0.7\textwidth,keepaspectratio=true]{./img/ergebnisse/fluoreszenz_assay/binding_inset.pdf} % spektrum.pdf: 428x377 pixel, 72dpi, 15.10x13.30 cm, bb=0 0 428 377 \caption[Häminbindung im Fluoreszenz-Assay]{Häminbindung im Fluoreszenz-Assay; Aufgetragen sind die relative Intensität des Fluoreszenzsignals gegen die Häminkonzentration. Letztere wurde aus einer Absorptionsmessung mit Hilfe des Extinktionskoeffizienten ermittelt. Das Inset zeigt eine halblogarithmische Auftragung der Daten. In beiden Auftragungen zeigt sich das Erreichen der Sättigung.} - \label{fig:fluoreszenz_spektrum} + \label{fig:fluoreszenz_bindung} \end{figure} \clearpage \section{\ac{CD}-Spektroskopie} \label{sec:ergebnis_cd_spektroskopie} +Die Daten der \ac{CD}-Spektren von \ac{RTE1} mit und ohne Hämin stammen aus unterschiedlichen Präparationen, da während der Messung von \ac{RTE1} mit Hämin ein Defekt an der Küvette und später am Spektrometer auftrat, so dass die Probe mit \ac{RTE1} ohne Hämin aus derselben Präparation nicht mehr gemessen werden konnte. Zum Vergleich wurden daher Daten aus einer vorangegangenen Messung von \ac{RTE1} ohne Hämin unter identischen Bedingungen herangezogen. Abbildung \ref{fig:cd_spektrum} zeigt beide Spektren nach Abzug der Pufferspektren von den Rohdaten. + +\begin{figure}[htbp!] + \centering + \includegraphics[width=0.7\textwidth,keepaspectratio=true]{./img/ergebnisse/cd_spektroskopie/spektrum.pdf} + % spektrum.pdf: 548x427 pixel, 72dpi, 19.33x15.06 cm, bb=0 0 548 427 + \caption[\ac{CD}-Spektren von gereinigtem \ac{RTE1}]{\ac{CD}-Spektren von gereinigtem \ac{RTE1}; Die Daten stammen von unterschiedlichen Präparationen von \ac{RTE1}. \ac{RTE1} wurde in \SI{50}{\milli\Molar} Kaliumphosphat-Puffer mit \SI{0,003}{\percent} (w/v) Fos-Cholin 16${^\text{\textregistered}}$ gelöst. Die Proteinkonzentration betrug \SI{0,05}{\milli\gram\per\milli\liter}. Die Spektren wurden mit einer Auflösung von \SI{0,5}{\nano\meter} (\ac{RTE1} + Hämin) bzw. \SI{1}{\nano\meter} (\ac{RTE1}) und \SI{20}{\nano\meter\per\minute} aufgenommen. Es wurden jeweils 15 Spektren akkumuliert. Deutlich ist in beiden Fällen ein negativer Cotton-Effekt bei etwa \SI{207}{\nano\meter} zu erkennen, welcher charakteristisch für $\alpha$-helikale Strukturanteile ist. Der $\alpha$-helikale Anteil ist bei Häminzugabe deutlich erhöht. } + \label{fig:cd_spektrum} +\end{figure} + +Anhand der \ac{CD}-Spektren wurde eine Sekundärstrukturanalyse mit \textit{CONTINLL} \citep{provencher_estimation_1981} durchgeführt. Sie ergab für \ac{RTE1} ohne Häminzugabe einen Sekundärstrukturanteil von \SI{18}{\percent} $\alpha$-Helix, \SI{30}{\percent} $\beta$-Faltblatt, \SI{22}{\percent} $\beta$-Schleife und \SI{30}{\percent} \textit{Random Coil}. Bei Zugabe von Hämin ergibt die Strukturvorhersage Anteile von \SI{44}{\percent} $\alpha$-Helix, \SI{6}{\percent} $\beta$-Faltblatt, \SI{25}{\percent} $\beta$-Schleife und \SI{25}{\percent} \textit{Random Coil}. Es zeigt sich also ein höherer Anteil von $\alpha$-Helices und ein geringerer Anteil $\beta$-Faltblättern. + + + diff --git a/inc/material.tex b/inc/material.tex index 341bb13..53675ae 100644 --- a/inc/material.tex +++ b/inc/material.tex @@ -565,11 +565,10 @@ Wie beim photometrischen Bindungsnachweis erfolgte die Bestimmung der tatsächli \subsubsection{\acs{CD}-Spektroskopie} \label{sec:nachweis_haemin_bindung_cd} -Bei der \ac{CD}-Spektroskopie wird die Wechselwirkung von optisch aktiven Substanzen mit zirkular polarisiertem Licht zu analytischen Zwecken ausgenutzt. Eine links- und eine rechtszirkulare Lichwelle überlagern sich hierbei zu linear polarisiertem Licht. Optisch aktive Substanzen weisen für die links- bzw. rechtszirkulare Komponente unterschiedliche Absorptionskoeffizienten \textepsilon$_\text{L}$ und \textepsilon$_\text{R}$, wobei letztlich ihre Differenz $\Delta\varepsilon$ gemessen und als Elliptizität $\theta$ angegeben. $d$ sei die Schichtdicke und $c$ die Konzentration $c$ angegeben \citep{lottspeich_bioanalytik_2006}: +Bei der \ac{CD}-Spektroskopie wird die Wechselwirkung von optisch aktiven Substanzen mit zirkular polarisiertem Licht zu analytischen Zwecken ausgenutzt. Eine links- und eine rechtszirkulare Lichwelle überlagern sich hierbei zu linear polarisiertem Licht. Optisch aktive Substanzen weisen für die links- bzw. rechtszirkulare Komponente unterschiedliche Absorptionskoeffizienten $\epsilon_\text{L}$ und $\epsilon_\text{R}$, wobei letztlich ihre Differenz $\Delta\varepsilon$ gemessen und als Elliptizität $\theta$ angegeben. $d$ sei die Schichtdicke und $c$ die Konzentration $c$ angegeben \citep{lottspeich_bioanalytik_2006}: \begin{equation} \theta (\lambda) = \Delta\varepsilon \cdot c \cdot d \end{equation} -Die \ac{CD}-Spektroskopie kann für die Analyse von Proteinsekundärstrukturen eingesetzt werden. Hierbei werden Spektren im Bereich von \SIrange{160}{250}{\nano\meter} aufgenommen. Man macht sich dabei zu Nutze, dass in diesem Bereich die $n \rightarrow \pi^*$ bzw. $\pi \rightarrow \pi^*$-Übergänge der Peptidbindung liegen. Durch ihre Chiralität reagiert das \ac{CD}-Spektrum eines Peptids sehr empfindlich auf Änderungen der Sekundärstruktur \citep{lottspeich_bioanalytik_2006}. Die Messungen erfolgten in CD-Puffer bei einer Proteinkonzentration von \SI{0,5}{\milli\gram\per\milli\liter}. - +Die \ac{CD}-Spektroskopie kann für die Analyse von Proteinsekundärstrukturen eingesetzt werden. Hierbei werden Spektren im Bereich von \SIrange{160}{250}{\nano\meter} aufgenommen. Man macht sich dabei zu Nutze, dass in diesem Bereich die $n \rightarrow \pi^*$ bzw. $\pi \rightarrow \pi^*$-Übergänge der Peptidbindung liegen. Durch ihre Chiralität reagiert das \ac{CD}-Spektrum eines Peptids sehr empfindlich auf Änderungen der Sekundärstruktur \citep{lottspeich_bioanalytik_2006}. Die Messungen erfolgten in CD-Puffer bei einer Proteinkonzentration von \SI{0,05}{\milli\gram\per\milli\liter}. Von den Rohdaten wurde das Pufferspektrum abgezogen und unter Berücksichtigung von Konzentration und Molekulargewicht in molaren \ac{CD} ($\Delta\varepsilon$) umgerechnet.