From c2d8833d897d9a8e74a7e5a660267a43fc7627f7 Mon Sep 17 00:00:00 2001 From: "W. Trevor King" Date: Mon, 25 Jun 2012 19:28:48 -0400 Subject: [PATCH] Flesh out apparatus chapter. Move I27 figure in from cantilever/methods, because we introduce I27 in the polymer-synthesis section. Additions: * Discussion of the piezo portion of an AFM. * Discuss history of force spectroscopy. * Add entire text of polymer synthesis and sample preparation sections. * Lots of little things. --- src/apparatus/afm.tex | 29 +++++- src/apparatus/main.bib | 9 ++ src/apparatus/main.tex | 4 +- src/apparatus/polymer-synthesis.tex | 128 ++++++++++++++++++++++++++- src/apparatus/procedure.tex | 1 + src/apparatus/sample-preparation.tex | 36 ++++++++ src/cantilever/methods.tex | 9 -- 7 files changed, 202 insertions(+), 14 deletions(-) diff --git a/src/apparatus/afm.tex b/src/apparatus/afm.tex index 65b358e..ef6babb 100644 --- a/src/apparatus/afm.tex +++ b/src/apparatus/afm.tex @@ -6,14 +6,16 @@ Of the mechanical manipulation methods listed in availability of user-friendly commercial instruments. AFM has been employed on several types of biological macromolecules, mechanically unfolding proteins\citep{carrion-vazquez99a} and forcing structural -transitions in DNA\citep{rief99} and polysaccharides\citep{rief97a}. +transitions in DNA\citep{florin95,rief99} and +polysaccharides\citep{rief97a}. An AFM\index{AFM} uses a sharp tip integrated at the end of a cantilever to interact with the sample. Cantilever bending is measured by a laser reflected off the cantilever and incident on a position sensitive photodetector (\cref{fig:afm-schematic}). When the bending force constant of the cantilever is known\citep{levy02}, the -force applied to the sample can be calculated. +force applied to the sample can be calculated using Hooke's law +(\cref{eq:sawsim:hooke}). \begin{figure} \asyinclude{figures/schematic/afm}% @@ -24,6 +26,29 @@ force applied to the sample can be calculated. position sensitive photodetector.\label{fig:afm-schematic}} \end{figure} +The substrate is mounted on a three dimensional piezoelectric actuator +so that the tip may be positioned on the surface with sub-nanometer +resolution (although signal drift and piezo hysteresis can cause +larger errors in the positioning accuracy). Our tubular piezo has a +range of TODO in the horizontal directions and a range of TODO in the +vertical (\cref{fig:piezo-schematic}). + +\begin{figure} + \asyinclude{figures/schematic/piezo}% + \caption{Schematic of a tubular piezoelectric actuator. In our AFM, + the substrate is mounted on the top end of the tube, and the + bottom end is fixed to the microscope body. This allows the piezo + to control the relative position between the substrate and the AFM + cantilever. The electrodes are placed so radial electric fields + can be easily generated. These radial fields will cause the piezo + to expand or contract axially. The $z$ voltage causes the tube to + expand and contract uniformly in the axial direction. The $x$ and + $y$ voltages cause expansion on one side of the tube, and + contraction (because of the reversed polarity) on the other side + of the tube. This tilts the tube, shifting the sample + horizontally.\label{fig:piezo-schematic}} +\end{figure} + % really, AFM can do this ;) The forces that can be applied and measured with an AFM range from tens of piconewtons to hundreds of nanonewtons. The investigation of diff --git a/src/apparatus/main.bib b/src/apparatus/main.bib index d91d648..4ec8e72 100644 --- a/src/apparatus/main.bib +++ b/src/apparatus/main.bib @@ -12,3 +12,12 @@ year = 2012, url = "http://www.athenaes.com/I27OAFMReferenceProtein.php", } + +@misc{ agilent-sure2, + author = "{Agilent Technologies}", + title = "SURE 2 Supercompetent Cells", + year = 2012, + url = "http://www.chem.agilent.com/en-US/Search/Library/_layouts/Agilent/PublicationSummary.aspx?whid=72739&liid=6524" + % http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=663 + note = "Catalog #200152.", +} diff --git a/src/apparatus/main.tex b/src/apparatus/main.tex index 8f71073..a54b74e 100644 --- a/src/apparatus/main.tex +++ b/src/apparatus/main.tex @@ -8,8 +8,8 @@ outline the procedure for synthesizing protein chains (\cref{sec:polymer-synthesis}). With the groundwork out of the way, we will look at sample preparation (\cref{sec:sample-preparation}) and the velocity clamp force spectroscopy procedure -(\cref{sec:procedure}). Finally, we will give an executive summary of -cantilever calibration (\cref{sec:cantilever-calib:intro}). +(\cref{sec:procedure}). Finally, we will give a summary of cantilever +calibration (\cref{sec:cantilever-calib:intro}). Everything discussed in this chapter, with the possible exception of cantilever calibration, is fairly standard practice in the field of diff --git a/src/apparatus/polymer-synthesis.tex b/src/apparatus/polymer-synthesis.tex index d8276fb..6a4992a 100644 --- a/src/apparatus/polymer-synthesis.tex +++ b/src/apparatus/polymer-synthesis.tex @@ -1,4 +1,130 @@ \section{Protein Polymer Synthesis} \label{sec:polymer-synthesis} -TODO. +Early experiments in force spectroscopy involved native +titin\index{titin}\citep{rief97a}. Titin is a muscle protein involved +in passive elasticity (\cref{fig:skeletal-muscle}), so it is an ideal +subject when examining the effect of mechanical force\citep{labeit95}. +Titin is also interesting because, while it is one of the largest +known proteins, it is composed of a series of globular domains. When +\citet{rief97a} carried out their seminal unfolding experiment, the +observed a very charachteristic sawtooth as the domains unfolded (see +\cref{sec:procedure} for a discussion of these sawteeth). + +\begin{figure} + \includegraphics[width=0.7\textwidth]{figures/binary/skeletal_muscle}% + \caption{Biological role of titin\index{titin}. Moving clockwise + from the upper left you can see a bone/muscle group, a muscle + fiber, a myofibril, and a sarcomere. In the sarcomere, the white, + knobbly filaments are actin. The myosin bundles are blue, and the + titin linkers are red. When the muscle contracts, the myosin + heads walk up the actin filiaments, shortening the sarcomere. + When the muscle relaxes, the myosin heads release the actin + filimants and slide back, lengthening the sarcomere. Titin + functions as an entropic spring that keeps the myosin from falling + out of place during the passive, relaxed stage. This figure is + adapted from \citet{skeletal_muscle}.\label{fig:skeletal-muscle}} +\end{figure} + +Unfortunately, it is difficult to analyze the unfolding of native +titin, because the heterogenous globular domains make it hard to +attribute a particular subdomain to a partuclar unfolding event. +Unfolding a single domain is not feasable because the large radius of +curvature of an AFM tip ($\sim20\U{nm}$\citep{olympus-tr400psa}) +dwarfs the radius of a globular domain +($\sim2\U{nm}$\citep{improta96}). When such a large tip is so close +to the substrate, van der Waals forces and non-specific binding with +the surface dominate the tip-surface interaction. In order to +increase the tip-surface distance while preserving single molecule +analysis, \citet{carrion-vazquez99b} synthesized a protein composed of +eight repeats of immunoglobulin-like domain 27 (I27), one of the +globular domains from native titin (\cref{fig:I27}). Octameric I27 +produced using their procedure is now available +commercially\citep{athenaes-i27o}. +\nomenclature{I27}{Titin immunoglobulin-like 27} + +\begin{figure} + \includegraphics[width=2in]{figures/i27/1TIT} + \caption{I27, the immunoglobulin-like domain 27 from human titin + (\href{http://dx.doi.org/10.2210/pdb1tit/pdb}{PDB ID: 1TIT})% + \citep{improta96}. + Figure generated with \citetalias{pymol}. + \label{fig:I27}} +\end{figure} + +Synthetic proteins are generally produced by creating a plasmid coding +for the target protein, inserting the plasmid in a bacteria, waiting +while the bacteria produce your protein, and then purifying your +proteins from the resulting culture. In this case, +\citet{carrion-vazquez99b} extracted messenger RNA coding for titin +from human cardiac tissue\cite{rief97a}, and used reverse +transcriptase to generate a complementary DNA (cDNA) library from +human cardiac muscle messenger RNA. This cDNA is then amplified using +the polymerase chain reaction (PCR), with special primers that allow +you to splice the resulting cDNA into a plasmid (which ends up with +one I27). Then they ran another PCR on the plasmid, linearized the +plasmid with two restriction enzymes, and grafted two I27-containing +sections together to form a new plasmid (now with two I27s, +\cref{fig:plasmid}). Another PCR-split-join cycle produced a plasmid +with four I27s, and a final cycle produced a plasmid with eight. The +eventual plasmid vector has the eight I27s and a host-specific +promoter that causes the bacteria to produce large quantities of I27. +The exact structure of the generated octamer +is\citep{carrion-vazquez99b} +\nomenclature{cDNA}{Complementary DNA} +\nomenclature{PCR}{Polymerase chain reaction} + +\begin{center} + Met-Arg-Gly-Ser-(His)$_6$-Gly-Ser-(I27-Arg-Ser)$_7$-I27-\ldots-Cys-Cys + \label{eq:I27} +\end{center} + +\begin{figure} + \includegraphics[width=0.9\textwidth]{figures/binary/kempe85-fig2}% + \caption{Example of gene duplication via plasmid splicing (Figure~2 + from \citet{kempe85}). \citet{kempe85} use a different gene, but + some of the restriction enzymes are shared with + \citet{carrion-vazquez99b}. The overall approach is + identical.\label{fig:plasmid}} +\end{figure} + +The plasmid is then transformed into the host, usually +\species{Escherichia coli}\citep{carrion-vazquez99b,bartels03,ma10} or +a proprietary equivalent such as Agilent's SURE 2 Supercompetent +Cells\citep{agilent-sure2,carrion-vazquez00}. The infected cells are +cultured to express the protein. +\nomenclature{Bacterial transformation}{The process by which bacterial + cells take up exogenous DNA molecules} +\nomenclature{Exogenous DNA}{DNA that is outside of a cell} + +The octamer is then purified from the culture using immobilized metal +ion affinity chromatography (IMAC), where the His-tagged end of the +octamer covalently bonds to a metal ion that is bound to the column +media (e.g. Ni-NTA\index{Ni-NTA} coated +beads)\cite{carrion-vazquez00,bartels03,ma10}. Once the rest of the +broth has been washed out of the chromatography column, the octamer is +eluted via either another molecule which competes for the metal +ions\citep{ma10} or by changing the pH so the octamer is less +attracted to the metal ion. +\nomenclature{IMAC}{Immobilized metal ion affinity chromatography} +\nomenclature{Ni-NTA}{Nickle nitrilotriacetic acid} + +\nomenclature{Ala}{Alanine, an amino acid} +\nomenclature{Arg}{Arginine, an amino acid} +\nomenclature{Asn}{Asparagine, an amino acid} +\nomenclature{Asp}{Aspartic acid, an amino acid} +\nomenclature{Cys}{Cystine, an amino acid} +\nomenclature{Glu}{Glutamine, an amino acid} +\nomenclature{Gly}{Glycine, an amino acid} +\nomenclature{His}{Histidine, an amino acid} +\nomenclature{Ile}{Isoleucine, an amino acid} +\nomenclature{Leu}{Leucine, an amino acid} +\nomenclature{Lys}{Lysine, an amino acid} +\nomenclature{Met}{Methionine, an amino acid} +\nomenclature{Phe}{Phenylalanine, an amino acid} +\nomenclature{Pro}{Proline, an amino acid} +\nomenclature{Ser}{Serine, an amino acid} +\nomenclature{Thr}{Threonine, an amino acid} +\nomenclature{Trp}{Tryptophan, an amino acid} +\nomenclature{Tyr}{Tyrosine, an amino acid} +\nomenclature{Val}{Valine, an amino acid} diff --git a/src/apparatus/procedure.tex b/src/apparatus/procedure.tex index e1927f3..adefad3 100644 --- a/src/apparatus/procedure.tex +++ b/src/apparatus/procedure.tex @@ -38,6 +38,7 @@ multi-domain test proteins. % \hspace{.25in}% \subfloat[][]{\asyinclude{figures/expt-sawtooth/expt-sawtooth}% \label{fig:expt-sawtooth}} + % Possibly use carrion-vazquez00 figure 2 to show scale of afm tip \caption{(a) Schematic of the experimental setup for mechanical unfolding of proteins using an AFM (not to scale). An experiment starts with the tip in contact with the substrate surface, which diff --git a/src/apparatus/sample-preparation.tex b/src/apparatus/sample-preparation.tex index 7b66dba..064efcb 100644 --- a/src/apparatus/sample-preparation.tex +++ b/src/apparatus/sample-preparation.tex @@ -1,3 +1,39 @@ \section{Sample Preparation} \label{sec:sample-preparation} +In mechanical unfolding experiments, one end of the protein is bound +to a substrate and the other binds to the AFM tip. This allows you to +stretch the protein by increasing the tip-substrate distance using the +piezo. A common approach is to synthesize proteins with cystine +residues on one end (\cref{eq:I27}) and allow the cystines to bind to +a gold surface\citep{carrion-vazquez99b,carrion-vazquez00,ulman96}. + +We prepare gold-surfaces by sputtering gold onto freshly cleaved mica +sheets in a vacuum. The mica keeps the gold surface protected from +contamination until it is needed. In order to mount the gold on our +AFM, we glue glass coverslips to the gold using a two part epoxy +(TODO). Instead of using mica to protect the gold surface, some labs +evaporate the gold directly onto the coverslips immediately before +running an experiment\citep{carrion-vazquez99b}. + +When it is time to deposit proteins on the surface, we peel a +coverslip off the gold-coated mica, exposing the gold surface that had +previously been attached to the mica. We dispense $5\U{$\mu$L}$ of +I27 solution ($65\U{g/$\mu$L}$) on the freshly-exposed gold, followed +by $5\U{$\mu$L}$ of phosphate buffered saline (PBS). We allow the +protein to bind to the gold surface for 30 mintues and then load the +coated coverslips into our AFM fluid cell. There are a number of +similar PBS recipes in common +use\citep{florin95,carrion-vazquez00,lo01,brockwell02}, but our PBS is +diluted from 10x PBS stock composed of $1260\U{mM}$ NaCl, $72\U{mM}$ +\diNaHPO, and $30\U{mM}$ \NadiHPO\citep{chyan04}. +\index{Phosphate buffered saline (PBS)} +\nomenclature{PBS}{Phosphate buffered saline} + +As an alternative to binding proteins to gold, others have used +EGTA\citep{kellermayer03}, +Ni-NTA\citep{schmidt02,itoh04,sakaki05,berkemeier11}, or silanized +glass\citep{sundberg03,ma10}. Some groups have also functionalized +the cantilever tips, with Ni-NTA being the most +popular\cite{schmitt00}. +\nomenclature{EGTA}{Ethylene glycol tetraacetic acid} diff --git a/src/cantilever/methods.tex b/src/cantilever/methods.tex index 83f100b..fe8830f 100644 --- a/src/cantilever/methods.tex +++ b/src/cantilever/methods.tex @@ -15,15 +15,6 @@ intermediate state followed by Bell-model escape to the unfolded state\citep{marszalek99}, although there is not yet a consensus of the presense of the proposed intermediate\citep{TODO}. -\begin{figure} - \includegraphics[width=2in]{figures/i27/1TIT} - \caption{I27, the immunoglobulin-like domain 27 from human Titin - (\href{http://dx.doi.org/10.2210/pdb1tit/pdb}{PDB ID: 1TIT})% - \citep{improta96}. - Figure generated with \citetalias{pymol}. - \label{fig:I27}} -\end{figure} - The I27 octamers were stored in a TODO buffer solution. Mechanical unfolding experiments were carried out on I27 octomers -- 2.26.2