getting down and dirty with detergents: quantitation, screening, and synthesis
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Getting down and dirty with detergents: quantitation, screening, and synthesis . 1 Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 2 Department of Chemistry, University of Wisconsin, Madison, WI 57306. - PowerPoint PPT PresentationTRANSCRIPT
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” WorkshopNational Institutes of Health
Bethesda, MDApril 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 wallPeriplasm
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
Current Statistics
Wes
tern
(ant
i-his)
Molecular Range of Expressed Membrane Proteins
100 75
50
30
15
Membrane Protein Production in Rhodobacter
Detergent Quantitation Detergent Screening Glycotripod amphiphiles
Enabling Technology/Reagent Short Story
Origin: Production core
Investigators:Chris KorsNick Impellitteri
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:
• time consuming• expensive• cumbersome
Detergent Quantitation Detergent ExchangeTHIN LAYER CHROMATOGRAPHY
– 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.
Average Exchange (%)TLC MS
Dialysis
On-column
69 18
98 2
49 15
97 0.3
Detergent Quantitation Detergent Screening Glycotripod amphiphiles
Enabling Technology/Reagent Short Story
Origin: Protein production core and detergent synthesis efforts
Investigators:Marc WanderAaron BowlingDeborah Hanson
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 issuesin 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.
• Maltosides tend to exhibit mild stabilizing characteristics (Levels 1 – 2).
• Glucosides are harsh, and dismantle the photosynthetic complex (Levels 3 – 5). • N-oxide groups display even harsher characteristics and are capable of dismantling the reaction center (Levels 3 – 6).
There are no correlations between:• Carbon chain length and protein stabilization.
• CMC and extraction/stabilization.
• Molecular weight and extraction/stabilization.
• Ionic nature and extraction/stabilization.
Some trends/patterns include:
How is this categorization planned to be used?
NEWProtein
Level 1Detergent
Level 2Detergent
Level 3Detergent
Level 4Detergent
Level 5Detergent
Level 6Detergent
Investigate a few detergent categories…
Examine 28 Level 1 Detergents
Examine 36 Level 2 Detergents
Examine 16Level 3 Detergents
Examine 9Level 4 Detergents
Examine 19Level 5 Detergents
Examine 9Level 6 Detergents
Determine which level detergent works best…
Explore only detergents in the desired class…
Examine 128 Detergents
Investigate ALL detergents…
NEWProtein
METHODMETHOD
1)
2)
…too time/material consuming!!
Detergent Quantitation Detergent Screening Glycotripod amphiphiles
Enabling Technology/Reagent Short Story
Origin: Detergent design and synthesis efforts
Investigators:Pil Seok ChaeSam Gellman
Application:Membrane protein solubilization, stabilization, and crystallization.
Design and Synthesis of Tripod Amphiphiles
a. Good solubility in aqueous media
b. Form micelles to extract the membrane proteins
Design of tripod amphiphiles
Synthesis of tripod amphiphiles
Exquisite balance between hydrophobicity and hydrophilicity
b. Scalable synthesis ( > 1.0 g): short steps and high yield
a. Facile structural modification by synthetic methods
c. High purity ( > 95 %) for reliable and definite results
c. Mild without denaturing of membrane protein complexes 8- Nonionic & zwitterionic amphiphiles vs. ionic amphiphiles
Quite efficient synthetic methods should be employed
Primary Detergents for Crystals of Membrane Proteins
Tripod amphiphile variants are being synthesized and evaluated
- +
HNO
NO
Hydrophobic moieties
Hydrophilic moieties
Nonionic ( glucose , maltose )Zwitterionic ( N- oxide )
O
O O
HOHO
OH
OH
OC12H25HOOH
HO
HO O
OH
OC8H17HOOH
Linear alkyl groups Nonlinear groups (aryl, cycloalkyl)
R
O
NH
O OHO
OH
HO OH
ONH
O OH
OHO
O
O
HO
HOOH
OH
OHOH
ONH
O
O
O
O OH
OOH
OOHOH
HO
OH
OHHO
HOOHOH
OH
ONH
OO O
OH OOH
HOOHOH
HOOH
ONH
OO OOH
HOO OHOH
HO
O O
OHO
OH O OH
OH
HO
OHHO OHOH
A five glycotripod amphiphile series
1
2
3
4
5
glucose
diglucose
triglucose
maltose
dimaltose
insoluble
high CMC
poor disruption
high CMC
poor disruption
TPA-2 rivals DDM for solubilization yieldmicelles are highly stabilizing (gentle)
Tripod versions of molecules 2 and 4 are superior to monopod version (tail replaced with C-12)
Saturated ring(TPA-2-S)
TPA-2-S is superior to DDM in this system.
Structural diversity among amphiphiles that efficiently extract and stabilize photosynthetic complexes from native membranes of R. capsulatus
O
NH
O
O
OH
OHO
OHHO
OHO
HO
OH
HO
I
O
NH
O
O
OH
OHO
OHHO
OHO
HO
OH
HO
O OO
HO
OHHO
OHO
HO
OHHO
NH
O
Note: Heavy Atom Incorporation
Note: Maltose Headgroup and Cyclization of Tripod Substituents
Note: Two Rings in Tripod
Tripod amphiphiles: selected recent developments
Acknowledgements
Program Project Members
Argonne National Lab– Deborah Hanson– Marc Wander– Aaron Bowling– Chris Kors– Nicholas Impelliterri
University of Wisconsin– Sam Gellman– Pil Seok Chae– Melissa Boersma
Outside Collaborations
University of Illinois – Chicago– Alex Schilling
Funding– NIH Roadmap Grant
• PO1 GM075913