Research In the Van Vranken Group

Applications of Combinatorial Libraries to the Discovery of Chemically Reactive Peptide Tags

Christopher J. McGee University of California, Irvine

Pro-fluorophore peptide protein of interest

Medicine is Linked To Understanding Protein Function

Genomics

40% of our proteins cannot be assigned a likely function by homology to other organisms Proteomics 80 % of our drug arsenal Targets proteins
(1,065 drugs out of 1,357)

Nature Reviews Drug Discovery. 2006, 5, 992996

Its Hard To Understand What You Cant See

Cell

s Dead cells cant convey dynamic character

Fluorescein

Direct visualization of the proteome: immunofluorescent labeling (dead Cells) bioorthogonal fluorescent labeling (live cells)

GFP Tags Allow Visualization in Living Cells

MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICTTGKLPVPWPTLVTTFSYGVQCFSRYP DHMKQHDFFKSAMPEGYVQERTIFYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNV Genetic engineering: add gene for GFP to gene for Protein of Interest YIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLEFVTA ARITHGMDELYK Introduce DNA into cell

Cell transcribes/translates protein+GFP, glued together

protein gene GFP gene

PROTEIN

Green Fluorescent Protein 238 Total Amino Acids

RNA

Cell

Aequorea victoria

t1/2 = ~3 min
Tsien. Annu. Rev. Biochem. 1998

t1/2 = 2080 min

GFP Reveals Location and Speed of HIV Transmission

H.I.V infected T cells expressing Gag protein fused to GFP

s We can see where a protein originated, where it went, how, and how fast s This mechanism of HIV-1 transmission could be important to its pathogenicity and may open new avenues for drug targets
Nat. Cell Biol. 2008 10, 2119

The Large Size of GFP Tags Limits Applications

sTags should NOT perturb the native folding, localization, or function of the POI s Many Human proteins are likely too small to study with fluorescent proteins Size of GFP 238 Amino Acds

relative frequency

Think this tracking device will impact the Bees behavior? Sequence length (from human proteome)

At best, you can shave 11 residues off the 238 in GFP, and still get fluorescence

Our Group Sought Smaller Fluorogenic Tags

s Dimerization and oxidation of Trps in peptides generates a highly fluorescent chromopohore

Can this be extended to biologically relevant peptides ?

J. Am. Chem. Soc. 1996, 118, 1225-1226.

Sequence-dependent fluorogenes in peptides is too slow for cellular labeling

pH 7.4 buffer 40 C

i) pick beads ii) cleave & sequence

Ala-Lys-Pro-Trp-Gly-Gly-Asp-Ala

J. Am. Chem. Soc. 2004, 126, 550-556.

OBOC Library Beads Contain Trillions of the Same Peptide

100 M

Amine Functionalize PEG Tails

1013 Amine Tails

TentaGel-HL-NH2 Resin Beads Bead Interior PEG Grafted to Polystyrene

= 320 pmol peptide per single bead

Each Bead = 1013 identical peptide molecules

Ala-Lys-Pro-Trp-Gly-Gly-Asp-Ala Ala-Lys-Pro-Trp-Gly-Gly-Asp-Ala Ala-Lys-Pro-Trp-Gly-Gly-Asp-Ala Ala-Lys-Pro-Trp-Gly-Gly-Asp-Ala

Peptide Tag That Binds Arsenic Containing Fluorescein

s Arsenic has high affinity for proteins containing proximal cysteines - toxic
Peptide Tag
i+4 i+5 S SH SH SH SH i+1 i S S S

Pro-Fluorophore

Fluorescent complex

sSmall vicinal thiols form tighter complexes with trivalent arsenic than cellular thiols -antidote

FlAsH-EDT2

+ 2 EDT

Tag Sequence: WEAAAREACCRECCARA

s Engineer a peptide tag having cysteines with greater arsenic affinity than dithiols sFluorogenesis was a bonus

Tags are labeled in less than 1 hr with with 1 mM EDT and 1 mM BAL

Tag
Tsien, et al. Science 1998

pro -flourophore

CCXXCC = Best Motif Out of 10 Peptides Screened

Kdapp (pM) 1800 67 100 150 70 72 42 41 4 72 92000

original

improved

What about : CXCXCXC, CCXCXXC, CXCCXC .

~240 nM Kd
Tsien, et al. JACS 2002

s These binding constants are not relevant dithiols always used in cellular screening

CCXXCC WDCCPGCCK = T1 Best case scenario FlAsH labels 60% of expressed tags We would begin our search for improved FlAsH tags here with OBOC libraries

Mammalian Cell Screens Optimizes Tag System 6 Fold

F.A.C.S F.A.C.S

Dithiol Washes

370 million NIH 3T3 Cells 230 million NIH 3T3 Cells XXXCCPGCCXXX XXCCXXCCXX

T2= T3= T1=

Offers 6 fold better contrast than T1 FLNCCPGCCMEP CCPGCC HRWCCPGCCKTF Confirmed WDCCPGCCK

s Experimentally T2 has shown better contrast and is the preferred tag

sStill 16 less sensitive than GFP sStill have only looked at only a few motifs (CCXCC)

FLNCCPGCCMEPReAsH Proposed Structure Unsetteling

s s s Flattened arsines No interstrand hydrogen bonds As2 is tilted only 44.3 from alignment

Grslund Model

with the xanthene -system Has an edge to face interaction between Phe and the xanthene ring system Elements Of 2Structure One Might Expect
As1

As2

As1

As2

Conformational Search for FLNCCPGCCMEPReAsH

Arsenics were pyramidal Hydrogen bond between the Cys4 & Cys8 (i and i+4) s Phe in close proximity to the xanthene system, consistent with NOE data Differed from Grslund models peptide backbone Why am I showing your this ? Later, well model one of our FlAsH Tags

As2

As1

Our Model

Grslund Model

Tetracysteine-Biased OBOC Library May Hold New Tags

s A cysteine-rich library (Ac-X-X-X-X-X-X-X-X-K-M-TentaGel) s s Bias towards 4 cysteines per peptide

Cysteine-Containing OBOC Libraries Are Unprecedented

The problems caused by cysteine during synthesis screening and sequencing leads to nearly complete exclusion of cysteine from OBOC libraries

s s s s

Disulfide bonds are often undesired during screening , thus reducing agents must be used Free cysteine undergoes side reactions in Edmandegradation and cyanogen bromide cleavage, chemistry used in two common methods for direct sequecening Cysteine is difficult to distiguinsh from arginine in Edman Sequencing

Screening The First Cys-Biased Library With FlAsH

Hits [FlAsH] 10 nM 10 nM 100 nM 1000 nM Round 1 2 3 4

FL1 Ac-X8-KM-Tentagel ~6.5 milion beads X = 10 different Amino Acids FlAsH

35 28 8 Many

Relative Reactivity 100 X 10X Average

1.0 mM BAL, 1.0 M BME MCB pH 7.2, 0.1% TrX-100

s In cell labeling, equilibrium is established in less than 1 hour with 1 mM dithiol

High concentration of monothiol was necessary to catalyze equilibrium on beads

WDCCPGCCKMFlAsH WDCCPGCCKM

Isolating a Hit, Alkylatingw/Cysteines, and Cleaving Peptide Screening The Library Nanomolar conc. Of FlAsH

MS/MS

How Does MS/MS Afford the Peptide Sequence

Subpopulations of parent ions with a mobile proton at a different amide bonds

De Novo MS/MS Identifies the Sequence of Hit Beads

Spectra of Ac-CYCFCRCCK-Hsl (Nanoflow LC-MS/MS)

Hits With Exactly 4 Cysteines Reveal New Reactive Motifs

1st Round of 10 nm Hits CCXCXXC CXCCXXC CCCXXC

2nd Round of 10 nm Hits Tsien CCXXCC CCCXXXC CXCCXC

s 5 New reactive motifs s s Endogenous human proteins with these motifs could lead to offtarget labeling and toxicity s s

Looking for Elements of 2 New Motifs

CCWCSSCReAsH CYCCLSCReAsH CCCPGCReAsH

C N

C N

s Weak H bond between the N-acetyl carbonyl and the NH of Trp3 (i and i+3)

s H bond between the NH of Tyr2 and carbonyl of Cys7 ( i and i+5) s

The three new motifs are not richly structured (nor was CCPGCC) Making it difficult to to guess their apptitude as FlAsH Tags. Of course, we can find out through experimentation

Making Sure Tag Reactivity is Reproducible Off of the Bead

1-7

1-12

1-12

T1

1-13

Pursue spots that: Give brighter complexes than T2 2-10 Cannot bind through CCXXCC

T2

Solution Phase Fluorescence Assay Comparing FlAsH Tags

sOur best hit was only 3 times less fluorescent than the T2 sequence T2 state of the art FlAsH tag (optimized) sSolution phase has low [monothiol] in contrast to Our best hit (not optimized) our bead and cellulose screens

sWe would go on to screen another library with low [monothiol]. No hits better than 2-10

Best Peptide YCCLCCPCKM-COOH

Possible Motifs CCXCXXC, CXCCXC, CCXXCXC, CXXCCXC

Modeled

CCXCXXC FlAsH Has Good Attributes For A Tag

i and i +3 H bond between Cys2 and Cys5 = hairpin turn i and i +2 bond hydrogen betweenCys7 and Cys9 = turn Salt Bridge between Lys9 and FlAsH Carboxylate Phe nearing an edge to face interaction with xanthene system

YCCLCCPCKM CCXCXXC

Discovering The 2 Best Known FlAsH Tags In One Scree

Employing the post screen dithiol wash (mimicking Tsiens cell screen)

We successfully identified sequences containing both known FlAsH tag Motifs: CCPGCC and CCKXCC

FB1 FB2 FB6

CCCCKCCCKM CCPCCCYNKM WDCCPGCCKM

sDramatic improvements have seemed unlikely sIdentification of several different reactive peptides suggests off-target labeling/ FB1 & FB2 FB3 FB5 toxicity as an obstacle that will FB4 remain as long as arsenic is part FB6the system of

Accomplishments from Biarsenical - Tetracysteine Work

s Longest and most diverse OBOC derived peptides ever sequence with MS/MS s s First MS/MS analysis applied to any oligomer containing cysteine

Accomplishments from Biarsenical - Tetracysteine Work

s First method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries s Could identify new reactive tags for biarsenicals s Identifed endogenous human protein targets s Protocols viable to screen for all important cysteine s reactivity heavy metal sequestering s s

New Fluorophore-Peptide Complexes From 1,4-Dicarbonyls

Fluorescent Isoindole

ortho-phthalaldehyde can condenses with amines and either a thiol or cyanide anion to generate a fluorescent isoindole in aqueous buffers

OPA derivatives with increased stability and better fluorescence properties 3-benzoyl-2-quinolinecarboxaldehyde (BQCA) is optimal There have been no attempts to find short peptides with the right juxtaposition of lysine and cysteine side chains that can react with OPA derivatives

Screening A Cysteine-Rich Library with BQCA

s10 nM hits lost to trapping cycle on nano-LC (C18) 13 100 nM hits sequenced

nanoLC MS/MS sequencing trouble shooting

fused silica emitter

movable metal stage

damaged emitter tip

www.newobjective.com/electrospray/index.html

Confirm The Red Fluorescence is Legit

493 ( ex )

m ax

Fluorophore is a good candidate for argon laser excitation (max output 488 nM)

SPOT Screen of13 Hit Sequences Reveals Best BQCA Tag

A SPOT array was screened with 500nM BQCA, 10 mM BuNH2, 5 mM BME
1

25

sCXXXXXXXK is prevalent sCK is prevalant

CK and CXXXXXXXK Do Not Appear Ideal

Relative Fluorescence of Designed Peptides

Optimized Variant of VWCCACSKM Discovered

sBecause VWCCACSKM superior in the initial SPOT array comparison, we made a library of variants.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 VWC WC V C VW VWC VWC VWC VWC VWC C C C C C C C C C C C C

original 2x optimization

30

VWC AWC VA C VWA VWC VWC VWC VWC VWC VWC VY C V F C VHC LWC I WC VWC VWC

C C C C A C C C C C C C C C C C C

A A A A A C A A A A A A A A A A A

Ala Scan

Deletions

A A A A A A A

C C C C C C C C C C C C C A C C C C C C C C C C

S S S S S S S S S S S S S A S S S S S S S T S

K KM KM KM KM KM KM KM KM KM KM KM KM AM KA KM KM KM KM KM KM RM

Point Deletions

A A A A

C C C C C

S S S S S S

K K K K K K K

M M M M M M M M

Adding A Reversible Chelating Group Enhance Selectivity

Precedence for aryl boronic acids being selective for serine sidechains in presence of cellular sugars (diols)

Screening A Ladder Library with BQCA Boronic Ester

Built a mass ladder into the library

Post Screen Chemical degredation Avoided MS/MS Avoided

Cys*

Tyr

Ala

Trp

Accomplishments from BQCA Work

s Identified the first peptide tags that react selectively with 1,4-dicarbonyls to give fluorescent isoindoles

Ac-VWCCACSKM-TentaGel s Synthesized a novel boronic ester variant of BQCA, with the capacity to react with four peptide sidechains

s Have shown the simplicity of the traditional ladder method offers a simple alternative to MS/MS

Acknowledgements
Dave Van Vranken *, Gary Juskowiak, Sean Devine, Romas Kudirka, Denise Dunn, Chris Adams, Avi Khanna, Glenn Eldridge

DVV Labmates:

Pete Belshaw, Shane Lamos, Casey Krusemark, Alex Shaginian, Marissa Rosen, Adam Miller GAANN Fellowship, Chem Office, Students, B-field Crew

UW Madison Labmates:

John Greaves & Shirin Sorooshian

Ms. Bareman (4th Grd) Mr. Jaeyk (7th Grd) Mr. Melter (HS) Mom and Dad Amber Reilly

Teachers

Roomates :

Any Structural Trends Between Motifs ?

CCPGCC CCCPGC
(From Peptide 1-13)

C N C Bead Screen Suggests CCCPGC is better than CCPGCC Modeling suggests CCPGC is better

N +6.3 kcal/mol

CCPGCC appears less strained , but both lack hydrogen bonding

Solution Phase Studies With BQCA

Red precipitate in mintues

1 of 2 possibilities