Getting down and dirty with detergents: quantitation, screening, and synthesis Philip D. Laible 1 , Samuel H. Gellman 2 , Deborah K. Hanson 1 , Christopher A. Kors 1 , Pil Seok Chae 2 , and Marc J. Wander 1 1 Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 2 Department of Chemistry, University of Wisconsin, Madison, WI 57306 Protein Structure Initiative “Bottlenecks” Workshop National Institutes of Health Bethesda, MD April 16, 2008
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Getting down and dirty with detergents: quantitation, screening, and synthesis Philip D. Laible 1, Samuel H. Gellman 2, Deborah K. Hanson 1, Christopher.
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Getting down and dirty with detergents: quantitation, screening, and synthesis
Philip D. Laible1, Samuel H. Gellman2, Deborah K. Hanson1, Christopher A. Kors1, Pil Seok Chae2, and Marc J. Wander1
1Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439
2Department of Chemistry, University of Wisconsin, Madison, WI 57306
Protein Structure Initiative “Bottlenecks” Workshop
National Institutes of Health
Bethesda, MD
April 16, 2008
Membrane proteins: ultra important but difficult to study
Roughly 65:35 split between soluble and membrane-associated proteins in most genomes.
Cytoplasmic and periplasmic volume is 30 times greater than membrane volume inside a typical cell.
Membrane proteins are key to many processes and comprise the majority of drug targets.
Structural and functional studies are difficult as membrane proteins are hard to produce.
Relatively few structures.
NIH/DOE
Innermembrane
Cell wall
Periplasm
Outer membrane
Typical membrane protein production pipeline
Primary focus of Program Project
A strategy to produce membraneproteins for reagent and technology tests
Advantage of the Rhodobacter expression system: This organism can be engineered to provide coordinated synthesis of foreign membrane proteins with synthesis of new membrane into which they can be incorporated.
Invaginations of thecell membranefound in speciesof Rhodobacter
Model of Rhodobacter
cells underscoring key features
Electron micrographs of two Rhodobacter deletion strains
Laible et al., 2007
400 Rhodobacter expression constructs have been evaluated.
Overall Rhodobacter expression success is ~ 60%.
Genes representing entire membrane proteomes are being cloned into the Rhodobacter membrane protein expression system with 80% efficiency. Ligation-independent cloning enabled a significantly higher-throughput approach to the test for successful heterologous expression for this target set.
Efficiency of Cloning, Conjugation, and Expression
Application:Production of well characterized/defined samples used throughout program.
We sought to develop a fast, inexpensive, and quantitative protocol to:
• create defined and reproducible membrane protein-detergent samples for input into structural and functional studies.
• facilitate replacement of detergents used for the solubilization and purification of a membrane protein with a diverse range of detergents that could potentially be more conducive to downstream characterization and crystallization attempts.
Measuring detergent levels inmembrane protein samples
Since determination of the detergent and lipid content of membrane protein samples can be:
– Place chromatography paper and solvent in TLC tank.– Equilibrate for one hour. – Spot samples on TLC plate. – Place plate in sealed chamber, allow solvent migration.– Remove and thoroughly dry TLC plate.
IODINE VAPOR STAINING– Incubate desiccator in water bath (60C).– Add iodine crystals.– Seal and stain for no more than 15 minutes.
SCANNING AND QUANTIFICATION– Immediately scan plate.– Quantification of spot intensities.
CONCENTRATE
ON-COLUMN
Samples bound to column, washed with 1,
5, 10, or 20 column volumes (CV) of
replacement detergent buffer, and eluted.
DIALYSIS
Samples dialyzed for 1, 2, 5, or 7 days
Input: PURE PROTEIN1] Rhodobacter sphaeriodes Reaction Center (RC)2] Escherichia coli protein APC809 (thiol:disulfide interchange protein)
DETERGENTEXCHANGE
All detergents, except Triton X-100 and C8E4, displayed unique Rf values, which were not altered when the detergents were run as a mixture in the same lane.
A detergent “ladder” (L) was created to aid in the identification of detergent spots.
Visualizing Detergent ‘Spots’ on TLC Plates
Detergents and Detergent Ladder
PurificationDetergent
ReplacementDetergent
Example TLC Plate
Analysis of detergents as PDCs had no effect on expected Rf values as well (similar results obtained with other detergents).
Detergents and Detection Limits
For all detergents surveyed:
Linear standard curves were obtained.
Detection limit spanned well below both the CMC and the concentrations of the detergents in the buffers used in this study.
Samples of a wide range of concentrations were run on TLC plates and then quantified in order to determine the range of detection limits for each detergent.
Detergent Exchange by Dialysis is Incomplete
Dialysis NEVER allowed for complete detergent exchange; substantial residual amounts of purification detergent (LDAO) remained.
Amount of residual purification detergent scaled proportionally with CMC of replacement detergent.
– OG (highest CMC of all the exchanging detergent) yielded ONLY 50 % exchange after 7 days.
– Triton X-100 (lowest CMC of all the exchanging detergents) yielded 87% exchange after 7 days.
On-Column Detergent Exchange is Quantitative
On-column detergent exchange was faster, more definitive and reproducible compared to dialysis for ALL detergents tested.
All detergents but one were able to replace 100% of the purification detergent after washing with only 5 column volumes.
Experimental Details for Sample Analysis with MS
Don’t need state-of-the-art Mass Spec(although we used an LTQ-FT)
Poroshell 300SB-C3 column Water/Acetonitrile Gradient Injected 1 µl sample Each detergent had a unique retention time Generated standard curve using peak areas Limited range of concentrations where response is
linear
TLC results confirmed with Mass Spectrometry
Cost Comparison
Mass Spectometer:– Need efficient access; if not, acquisition costs astounding.– Method development alone can cost hundreds of dollars.– Individual sample runs are at least $50 (possibly > $100).
TLC with Iodine Vapor Staining:– Portable with minimal costs (once a desiccator, TLC tank, hot water bath, and a scanner were obtained). – The costs involved for chemicals, TLC plates, and chromatography paper were less than 50 cents per sample.
Application:Discovery of new, generally useful, surfactants. Categorize known sets of detergents to make work with them less trial and error.
Detergent Selection
Detergent properties and micelle properties influence:
Yield of protein extracted from the lipid bilayer
Protein stability
Quantity and type of native lipids which are co-extracted with integral or membrane-associated proteins
Ultimately, functional properties and structural integrity
Crystallization propensity; thus, the solubilizing detergent may have to be exchanged before trials are initiated
a super-critical step in a purification scheme
Zhang et al, 2003
The ranking system focuses upon two important initial issues
in membrane protein purification:
The Detergent Screening Protocol
• Solubilization – tests ability of the detergent to disrupt the lipid bilayer and extract protein
• Stabilization – tests the ability of micelles of a detergent to stabilize the protein once removed from the membrane
• LHII is very stable, and therefore removed from our starting material
• LHI is very fragile and readily falls apart
• RCs are intermediate
The Screening Protocol: A Closer Look
Weak StrongWeak detergents extract complexes with LHI intact
Intermediate detergents break down LHI, RCs remain intact
Strong detergents break down LHI and RCs
Rhodobacter capsulatus strainutilized lacks LHII
LHI
RC
HT
Standardized protocol amenable to automation
Screening commences with homogenized Rhodobacter capsulatus membranes and proceeds on a relatively small scale in order to maximize the number of detergents that can be examined.
The Ranking System in Action
Strong surfactantLDAO
Intermediate surfactantTriton X-100, OG
Weak surfactantDDM, HEGA-11, CHAPS
Level 5Detergent
Level 3Detergent
Level 1Detergent
Weak Strong
+
Summary of Results
A total of 128 detergents have been investigated(e.g. Anatrace, Cognis, Sigma, Avanti Polar Lipids)
• Most detergents tested have a carbon chain length between 7 and 12 (broad range of extraction yield).
• Detergents with chain lengths <7 carbons have more consistent extraction success. Detergents with >12 carbons tend to have poor extraction.