Dissertation Talk7

Research In the Van Vranken Group

Christopher J. McGeeUniversity of California, Irvine

protein of interest

peptide

Pro-fluorophore

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

Medicine is Linked To Understanding Protein Function

40% of our proteins cannot be assigned a likely function

by homology to other organisms

80 % of our drug arsenal Targets proteins

(1,065 drugs out of 1,357)

Genomics

Proteomics

Nature Reviews Drug Discovery. 2006, 5, 992–996

It’s Hard To Understand What You Can’t See

Cell

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

n Dead cells can’t convey dynamic character

Fluorescein

GFP “Tags” Allow Visualization in Living Cells

Green Fluorescent Protein

MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLEFVTAARITHGMDELYK

238 Total Amino Acids

Aequorea victoria

Genetic engineering: add gene for GFP to gene for Protein of Interest

Introduce DNA into cell

Cell transcribes/translates protein+GFP, glued together

protein gene GFP gene

RNA

PROTEIN

Cell

t1/2 = ~3 min t1/2 = 20–80 minTsien. Annu. Rev. Biochem. 1998

Nat. Cell Biol. 2008 10, 211–9

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

GFP Reveals Location and Speed of HIV Transmission

n This mechanism of HIV-1 transmission could be important to its pathogenicity and may open new avenues for drug targets

n We can see where a protein originated, where it went, how, and how fast

The Large Size of GFP Tags Limits Applicationsn Tags should NOT perturb the native folding, localization, or function of the

POI n Many Human proteins are likely too small to study with fluorescent proteins

Size of GFP238 Amino Acds

rela

tive

freq

uenc

y

Sequence length (from human proteome)

Think this tracking device will impact the Bee’s behavior?

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

pH 7.4 buffer i) pick beads

ii) cleave &sequence

40 ｡C

n Dimerization and oxidation of Trp’s in peptides generates a highly fluorescent chromopohore

Can this be extended to biologically relevant peptides ?

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

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

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

Our Group Sought Smaller Fluorogenic Tags

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

100 µM

TentaGel-HL-NH2Resin Beads

Bead Interior PEG Grafted to Polystyrene

Amine Functionalize PEG Tails

OBOC Library Beads Contain Trillions of the Same Peptide

1013 Amine Tails

= 320 pmol peptide per single bead

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

Each Bead =

1013 identical peptide molecules

n Fluorogenesis was a bonus

n Arsenic has high affinity for proteins containing proximal cysteines - toxic

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

n Engineer a peptide tag having cysteines with greater arsenic affinity than dithiols

Peptide Tag That Binds Arsenic Containing Fluorescein

Tsien, et al. Science 1998

SH SH

SHSH

ii+1

i+4 i+5

S

S

S

S

FlAsH-EDT2 + 2 EDT

WEAAAREACCRECCARATag Sequence:

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

Peptide Tag Pro-Fluorophore Fluorescentcomplex

Tagpro -flourophore

CCXXCC = Best Motif Out of 10 Peptides Screened

We would begin our search for improved FlAsH tags here –with OBOC libraries

Tsien, et al. JACS 2002

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

180067

100

707242

472

92000

150

41

Kdapp(pM)

CCXXCCWDCCPGCCK = T1

original

improved

~240 nM Kd

Best case scenario FlAsH labels 60% of expressed tags

What about :CXCXCXC, CCXCXXC, CXCCXC ….

Mammalian Cell Screens Optimizes Tag System 6 Fold

F.A.C.S Dithiol

Washes

230 million NIH 3T3 CellsXXCCXXCCXX

CCPGCC Confirmed

F.A.C.S Dithiol

Washes

370 million NIH 3T3 CellsXXXCCPGCCXXX

T2= FLNCCPGCCMEP T3= HRWCCPGCCKTF

T1= WDCCPGCCK

Offers 6 fold better contrast than T1

n Still 16 less sensitive than GFPn Still have only looked at only a few motifs (CCXCC)

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

FLNCCPGCCMEP•ReAsH Proposed Structure Unsetteling

As1

As2

As1As2

n Flattened arsinesn No interstrand hydrogen bondsn As2 is tilted only 44.3° from

alignment with the xanthene π-system Has an edge to face interaction between Phe and the xanthene ring system

Elements Of 2° Structure One Might Expect

Gräslund Model

Conformational Search for FLNCCPGCCMEP•ReAsH

Hydrogen bond between the Cys4 & Cys8 (i and i+4)

n Phe in close proximity to the xanthene system, consistent with NOE data

As1

As2

Arsenics were pyramidal

Differed from Gräslund model’s peptide backbone

Gräslund Model Our Model

Why am I showing your this ? Later, we’ll model one of our FlAsH Tags

n A cysteine-rich library (Ac-X-X-X-X-X-X-X-X-K-M-TentaGel)

n Bias towards 4 cysteines per peptide

Tetracysteine-Biased OBOC Library May Hold New Tags

Cysteine-Containing OBOC Libraries Are Unprecedented

n Disulfide bonds are often undesired during screening , thus reducing agents must be used

n Free cysteine undergoes side reactions in Edman degradation and cyanogen bromide cleavage, chemistry used in two common methods for direct sequecening

n Cysteine is difficult to distiguinsh from arginine in Edman Sequencing

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

Hits [FlAsH] Round Relative Reactivity

35 10 nM 1 100 X28 10 nM 28 100 nM 3 10X

Many 1000 nM 4 Average

Screening The First Cys-Biased Library With FlAsH

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

FlAsH

FL1Ac-X8-KM-Tentagel~6.5 milion beads

X = 10 different Amino Acids

n 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

WDCCPGCCKM•FlAsH

WDCCPGCCKM

Screening The Library w/ Nanomolar conc. Of FlAsHIsolating a Hit, Alkylating Cysteines, and Cleaving Peptide

MS/MS

How Does MS/MS Afford the Peptide Sequence

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

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

De Novo MS/MS Identifies the Sequence of ‘Hit’ Beads

1st Round of 10 nm Hits 2nd Round of 10 nm Hits

Hits With Exactly 4 Cysteines Reveal New Reactive Motifs

CCXCXXCCXCCXXC

CCCXXC

Tsien CCXXCCCCCXXXC

CXCCXC

n 5 New reactive motifs

n Endogenous human proteins with these motifs could lead to off-target labeling and toxicity

C

N

CCWCSSC•ReAsH CYCCLSC•ReAsH

N C

Looking for Elements of 2° New Motifs

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

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

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

CCCPGC•ReAsH

N

C

Making Sure Tag Reactivity is Reproducible Off of the Bead

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

1-7

1-131-12 1-12

2-10 T2

T1

n Solution phase has low [monothiol] in contrast to our bead and cellulose screens

Solution Phase Fluorescence Assay Comparing FlAsH Tags

n Our best hit was only 3 times less fluorescent than the T2 sequence

T2 –state of the art FlAsH tag (optimized)

Our best hit (not optimized)

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

Best Peptide YCCLCCPCKM-COOH

CCXCXXC, CXCCXC,CCXXCXC, CXXCCXC

Possible Motifs

i and i +3 H bond between Cys2 and Cys5 = b hairpin turn i and i +2 bond hydrogen betweenCys7 and Cys9 = g turn

N

C

Salt Bridge between Lys9 and FlAsH Carboxylate Phe nearing an edge to face interaction with xanthene system

Y C C L C C P C K MC C X C X X C

Modeled CCXCXXC•FlAsH Has Good Attributes For A Tag

Discovering The 2 Best Known FlAsH Tags In One Screen

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

FB1 & FB2 FB3 FB4 FB5 FB6

FB1 CCCCKCCCKMFB2 CCPCCCYNKMFB6 WDCCPGCCKM

Employing the post screen dithiol wash (mimicking Tsien’s cell screen)

n Dramatic improvements have seemed unlikely n Identification of several different reactive peptides suggests off-target labeling/ toxicity as an obstacle that will remain as long as arsenic is part of the system

Accomplishments from Biarsenical-Tetracysteine Work

n Longest and most diverse OBOC derived peptides ever sequence with MS/MS

n First MS/MS analysis applied to any oligomer containing cysteine

n First method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries

Accomplishments from Biarsenical-Tetracysteine Work

n Could identify new reactive tags for biarsenicalsn Identifed endogenous human protein targetsn Protocols viable to screen for all important cysteine reactivity – heavy metal sequestering

New Fluorophore-Peptide Complexes From 1,4-Dicarbonyls

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

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

3-benzoyl-2-quinolinecarboxaldehyde (BQCA) is optimal

Fluorescent Isoindole

n 10 nM hits lost to trapping cycle on nano-LC (C18)

13 100 nM hits sequenced

Screening A Cysteine-Rich Library with BQCA

fused silica emitter

movable metal stagedamaged emitter tip www.newobjective.com/electrospray/index.html

nanoLC MS/MS sequencing trouble shooting

Confirm The Red Fluorescence is Legit

493 (λ max ex)

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

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

SPOT Screen of 13 Hit Sequences Reveals Best BQCA Tag

1

25

n CXXXXXXXK is prevalent

n CK is prevalant

CK and CXXXXXXXK Do Not Appear Ideal

Relative Fluorescence of Designed Peptides

n Because VWCCACSKM superior in the initial SPOT array comparison, we made a library of variants.

1 V W C C A C S K M2 W C C A C S K M3 V C C A C S K M4 V W C A C S K M5 V W C C C S K M6 V W C C A S K M7 V W C C A C K M8 V W C C A C S M9 V W C C A C S K

10 C C A C S K M11 C A C S K M12 A C S K M13 C S K M14 V W C C A C S K M15 A W C C A C S K M16 V A C C A C S K M17 V W A C A C S K M18 V W C A A C S K M19 V W C C C C S K M20 V W C C A A S K M21 V W C C A C A K M22 V W C C A C S A M23 V W C C A C S K A24 V Y C C A C S K M25 V F C C A C S K M26 V H C C A C S K M27 L W C C A C S K M28 I W C C A C S K M29 V W C C A C T K M30 V W C C A C S R M

Po

int

De

letio

ns

De

letio

ns

Ala

Sca

n

30

Optimized Variant of VWCCACSKM Discovered

2x optimization

original

Adding A Reversible Chelating Group – Enhance Selectivity

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

Built a mass ladder into the library

Post Screen Chemical degredation Avoided

MS/MS Avoided

Screening A Ladder Library with BQCA Boronic Ester

Cys* AlaTyr Trp

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

n Synthesized a novel boronic ester variant of BQCA, with the capacity to react with four peptide sidechains

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

Ac-VWCCACSKM-TentaGel

Accomplishments from BQCA Work

Acknowledgements

Roomates:Mom and DadAmber Reilly

DVV Labmates:Dave Van Vranken*, Gary Juskowiak, Sean Devine, Romas Kudirka,

Denise Dunn, Chris Adams, Avi Khanna, Glenn Eldridge

UW Madison Labmates:Pete Belshaw, Shane Lamos, Casey Krusemark, Alex Shaginian,

Marissa Rosen, Adam Miller

TeachersMs. Bareman (4th Grd) Mr. Jaeyk (7th Grd) Mr. Melter (HS)

GAANN Fellowship, Chem Office, Students, B-field Crew

John Greaves & Shirin Sorooshian

Any Structural Trends Between Motifs ?

CCPGCC CCCPGC

C

C

N

N

(From Peptide 1-13)

Bead Screen Suggests CCCPGC is better than CCPGCC

Modeling suggests CCPGC is better

CCPGCC appears less strained , but both lack hydrogen bonding

+6.3 kcal/mol

Red precipitate in mintues

Solution Phase Studies With BQCA

1 of 2 possibilities