From c81a8ac502e851e587a1ecdeab362739a7f19f7f Mon Sep 17 00:00:00 2001 From: "W. Trevor King" Date: Mon, 27 May 2013 08:56:30 -0400 Subject: [PATCH] salt/main.tex: Start to summarize my salt experiments --- src/local_cmmds.tex | 3 ++ src/root.tex | 2 + src/salt/main.tex | 91 +++++++++++++++++++++++++++++++++++++++++++++ 3 files changed, 96 insertions(+) create mode 100644 src/salt/main.tex diff --git a/src/local_cmmds.tex b/src/local_cmmds.tex index a8562fd..ae29751 100644 --- a/src/local_cmmds.tex +++ b/src/local_cmmds.tex @@ -144,10 +144,13 @@ % Chemicals \newcommand{\Ca}{Ca\textsuperscript{2+}} \newcommand{\CaCl}{CaCl\textsubscript{2}} +\newcommand{\Cl}{Cl\textsuperscript{-}} +\newcommand{\HCl}{HCl} \newcommand{\Na}{Na\textsuperscript{+}} \newcommand{\NaCl}{NaCl} \newcommand{\diNaHPO}{Na\textsubscript{2}HPO\textsubscript{4}} \newcommand{\NadiHPO}{NaH\textsubscript{2}PO\textsubscript{4}} +\newcommand{\NaOH}{NaOH} % Workaround for inline minted markup % http://code.google.com/p/minted/issues/detail?id=15 diff --git a/src/root.tex b/src/root.tex index 8de670f..a461de1 100644 --- a/src/root.tex +++ b/src/root.tex @@ -70,6 +70,7 @@ defaultpen(fontsize(10)); // match drexel-thesis's default 10pt font size \include{hooke/main} %\include{temperature/main} %\include{cantilever/main} +\include{salt/main} \include{future/main} \end{thesis} @@ -80,6 +81,7 @@ defaultpen(fontsize(10)); // match drexel-thesis's default 10pt font size pyafm/main,% calibcant/main,% hooke/main,% + salt/main,% packaging/main,% figures/main,% root} diff --git a/src/salt/main.tex b/src/salt/main.tex new file mode 100644 index 0000000..2445512 --- /dev/null +++ b/src/salt/main.tex @@ -0,0 +1,91 @@ +\chapter{The effect of ions on unfolding force} +\label{sec:salt} + +With the tools in place, it's time to do some science! As a simple +experiment to demonstrate the utility of the new stack, I ran a series +of velocity clamp unfolding experiments on I27 octomers in PBS +(\cref{sec:sample-preparation}). After a series of pulls in the +standard buffer, a buffer with additional sodium (from +\NaCl\citep{bdh-NaCl}) or calcium (from \CaCl\citep{fisher-CaCl}) +was flushed into the fluid cell. After the new buffer equilibrated, +unfolding experiments were continued. + +Previous research on the effect of ions on unfolding forces is small, +although earlier experimental work on Amyloid $\beta$ shows decreased +fibrillation with even small +\Ca\ concentrations\citep{chauhan97,itkin11}. The mechanism behind +this weaker bonding is unclear\citep{chauhan97,zhang06}, although +aspartic and glutamic acid groups tend to have a strong affinity for +cations while argenine has a strong affinity for the +\Cl\ anion\citep{friedman11}. Of these amino acids, only glutamic +acid occurs in the key hydrogen bond regions responsible for I27 +unfolding\citep{lu00b}. \NaCl\ has also been shown to decrease +hydrogen bonding\citep{zidar11}. + +We added $0.5\U{M}$ \CaCl\ to our standard PBS +(\cref{sec:sample-preparation}), which is much larger than +extracellular \Ca\ levels on the order of +$2\U{mM}$\citep{isaacs06,itkin11}. After mixing, both buffers were +adjusted with drops of \HCl\ and \NaOH\ as needed to reach a pH around +7.5 (7.42 for the PBS, and 7.60 for the \Ca-enhanced PBS). + +Unfolding experiments carried out on 2013-03-04 using our usual +procedure (\cref{sec:procedure}) yielded an unusual density of clean +unfolding curves\footnote{ + Experiments carried out using the same procedures throughout + February were much less successful. +}, +with 105 successful pulls concentrated in a two hour window. Of these +pulls, 37 were in the standard PBS and 68 were in the enhanced +\Ca\ buffer. Histograms of unfolding forces show decreased +unfolding forces in the \Ca\ buffer (\cref{fig:calcium:histogram}), +which is what we expect due to destabilized hydrogen bonding. + +\begin{figure} + \begin{center} + \includegraphics[width=0.9\textwidth]{figures/salt/2013-03-04-CaCl2} + \caption{I27 runs from 2013-03-04 with (red) and without (blue) an + extra $0.5\U{m}$ \Ca. Clockwise from the upper left, we have + the distance (in nm) between peaks, the unfolding force (in pN), + and example force curve, and a scatter plot of unfolding force + (in pN) versus the distance between peaks. All of the pulls + were taken with the same Olympus TR400-PSA cantilever with a + pulling speed of $1\U{$\mu$m/s}$. The green histogram drawn + over the unfolding force histograms is I27 unfolding data in PBS + with $5\U{mM}$ DTT from \citet{carrion-vazquez99b}, rescaled by + a factor of $\frac{1}{2}$ because they had more unfolding + events.\label{fig:calcium:histogram}} + \end{center} +\end{figure} +% +\nomenclature{DTT}{Dithiothreitol + (C\textsubscript{4}H\textsubscript{10}O\textsubscript{2}S\textsubscript{2}), + also known as Cleland's reagent\citep{cleland64}. It can be used to + reduce disulfide bonding in proteins.} + +Modeling I27 as a Bell-model unfolder, we can use \sawsim\ to find the +Bell parameters that best fit these experimental unfolding histograms +(\cref{sec:sawsim:rate:bell,sec:sawsim:results:fitting}). The results +in \cref{fig:calcium:valley} show that the best fit for standard PBS +was with $\Delta x_u = TODO\U{nm}$ and $k_{u0}=TODO\U{s$^{-1}$}$. In +the \Ca\ buffer, the best fit was with $\Delta x_u = TODO\U{nm}$ and +$k_{u0}=TODO\U{s$^{-1}$}$. + +\begin{figure} + \begin{center} + \subfloat[][]{% + \includegraphics[width=0.45\textwidth]{figures/salt/valley-PBS}% + \label{fig:calcium:valley:pbs}} + \subfloat[][]{% + \includegraphics[width=0.45\textwidth]{figures/salt/valley-PBS-CaCl2}% + \label{fig:calcium:valley:pbs-calcium}} + \caption{\protect\subref{fig:calcium:valley:pbs} Model fit quality + for the standard PBS unfolding histogram data shown in + \cref{fig:calcium:histogram}. + \protect\subref{fig:calcium:valley:pbs-calcium} Model fit + quality for the \Ca-enhanced PBS unfolding histogram data. The + best fit parameters occur when the Jensen--Shannon divergence is + minimized (at the bottom of these valleys, + \cref{sec:sawsim:results:fitting}).\label{fig:calcium:valley}} + \end{center} +\end{figure} -- 2.26.2