Supplementary Data

Malachite Green Mediates Homodimerization of Antibody VL Domains to Form a Fluorescent Ternary Complex with Singular Symmetric Interfaces. Chris Szent-Gyorgyi1, Robyn L. Stanfield2, Susan Andreko1, Alison Dempsey1, Mushtaq Ahmed1, Sarah Capek3, Alan S. Waggoner1,3, Ian A. Wilson2,4, and Marcel P. Bruchez1,3,5

1 - Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA 2 - Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 3 - Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA 4 - The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 5 - Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA

Contents Table S1. C2 symmetric 2:1 protein:ligand complexes in PDB (provided as separate Excel file). Table S2. Hydrogen bonds to waters in the AB interface of the unliganded L5* structure.

Figure S1. L5* derives from an atypical λ class germ line allele.

Figure S2. Characterization of liganded and unliganded L5*.

Figure S3. Quaternary structure comparison of VL/VL interfaces.

Figure S4. Contact analysis of unliganded and liganded L5* VL/VL complexes.

Figure S5. Features of L5* binding region.

Figure S6. Conformation of MG.

Figure S7. Spectral evidence that L5* ternary complex is the fluorescent form.

Figure S8. Thermodynamic parameters of binding MG to the linked dimer dL5* compared to other

protein/small molecule complexes.

Figure S9. Improved in vivo visualization and cytometry using dL5**. Supplementary Data References

S1

S2

(Supplementary Table S1 provided as separate Excel File)

Table S1. C2-symmetric 2:1 protein:ligand complexes in PDB. Public structural databases cannot be directly queried for protein homodimer/ligand complexes defined by a C2 symmetry axis inclusive of protein and ligand. Therefore, the PDB databank (http://www.rcsb.org/pdb, 81155 protein entries as of 01/22/2013) was used to identify all macromolecule records that contain only a single polypeptide species present in a two-chain structure and also contain one or more free ligands of MW 200–2000. A set of 10,243 such structures, each containing one or more of 4163 unique ligands, was identified and downloaded as an Excel file. The ligands associated with these structures were individually evaluated for C2 symmetry by eye using on-line PDB images; 243 C2-symmetric ligands of MW 200–2000 were identified. This 243 member ligand list was then used to search the entire PDB for associated protein records, again filtering for macromolecule records that contain only a single polypeptide species present in a two-chain structure, and also contain one or more free ligands of MW 200–2000. Because PDB searches based on ligand lists return information on other ligands associated with PDB IDs, 896 ligands were identified and downloaded with associated protein records as an Excel file. The 243 C2-symmetric ligand set and associated records were extracted, and simple linear polymers or ligands that contain coordinating metals were culled. PDB protein records associated with the remaining 179 C2-symmetric ligands were then evaluated by eye for co-complexes that display superposed C2 symmetry. The ligands and PDB records are presented in Table S1 as an Excel spreadsheet with live links to the PDB. Of the 179 C2-symmetric ligands, 52 were found in one or more PDB records to be members of a protein homodimer complex where proteins and ligands display a superposed C2 symmetry axis; 39 of these ligands are HIV protease inhibitors (average MW of 699.7). Other than the HIV protease complexes, our manual survey of the PDB identifies only 13 protein homodimer/ligand co-complexes with shared C2 symmetry; only 8 of these co-complexes contain C2-symmetric organic ligands related to biological function.

S3

L5* residue Water Distance

(Å)

A Y32 O# D 8 3.23

A N34 OD1 D 8 2.69

A N34 ND2# D 75 3.13

A I48 O D 20 2.73

A E50 N D 8 3.03

A Y55 OH# D 20 2.91

A Y55 OH# D 115 3.28

A S91 OG# D 35 2.49

A Y96 OH# D 55 2.68

B I48 O D 17 2.64

B Y55 OH# D 75 3.27

B Y55 OH# D 53 3.11

B Y55 OH# D 118 3.07

B Y96 OH D 55 2.72

Table S2. Hydrogen bonds to waters in the AB interface of the unliganded L5* structure. Atoms with (#) are also in van der Waals contact to MG in the liganded L5* structure. A, B and D are ‘chain’ descriptors listed in the PDB file.

S4

0

2

4

6

8

10

12

-4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1

Z-score

Comparison: Light chain Lambda class

QueryHuman

L5* IGL V7-46

1.5

Fre

quency

of

occ

urr

ence

Fig. S1. L5* derives from an atypical λ class germline allele. Relatedness of L5* [1,2] and λ IGL-V7-46 to the expressed repertoire of human λ class genes by Z-score. Plotted Z-scores are derived from standard deviations of a sequence’s identity to the λ class repertoire in the Kabat database [3]. L5* and its germline progenitor fall within an outlying distribution representing those 2% of λ sequences that diverge most from typical sequences in the λ repertoire.

S5

28470Da

+ MG

14170Da

– MG

L5*

(a)

(c)

C

N

V AL

20º

V AL V BL

(b)

L5 L5lysozyme lysozyme

T = 0 hrs T = 6 hrs

10

152025

37

kb

+ + ++ ___ _

Fig. S2. Characterization of liganded and unliganded L5*. (a) Size exclusion chromatography of L5*. 25 µl injections of ~1 mg/ml (~80 µM) liganded or unliganded protein were run on a Superdex S75 10/300 analytical gel filtration column. The molecular masses corresponding to L5* peaks were determined using a separate calibration chromatography run (data not shown). (b) Crosslinking of L5*. SDS PAGE gel stained with Brilliant Blue R-250 is shown as an unadjusted grayscale image. 40 µM L5* protein in the presence (+) or absence (-) of 50 µM MG-ester1 was cross-linked with 0.05 % glutaraldehyde in pH 5.0 buffer. Lysozyme is used as a control. MG-ester was used because the amine at the terminus of the MG-2p PEG linker is a target for cross-linking. Other cross-linking experiments at neutral pH and in the presence of CHAPSO or pluronic F-127 detergents failed to cross-link L5* under conditions where H6 FAP [1] readily crosslinks after 1 minute incubation. The partial crosslinking achieved only after prolonged incubation may reflect the dominance of intradomain crosslinks between Lys53 and Lys54 that prevent stable association of VL domains. (c) Reorientation of L5* VLs upon binding MG. Top, tube representations of superposed liganded (slate) and unliganded (salmon) VL A domains showing divergent main-chain paths at CDR3 (red in liganded VL). Val3 (red) and Val106 (green) Cα space-fill atoms are shown to mark respective N- and C-termini. A second loop at residues 39-43 (blue in liganded VL) also diverges in the liganded structure; the second loop contacts the second loop of a neighboring dimer in the crystal unit via an Ala43-Gly41 hydrogen bond, and thus this path divergence may be due to a crystallization artifact. Bottom, liganded and unliganded VL/VL dimer structures with the VL A domains superposed as previously, now seen in a top view. Relative to the unliganded dimer, the VL B domain of the liganded dimer is rotated towards the back by ~20º as shown, which corresponds to a separation of 8.5 Å for the Val106 Cα atoms.

S6

1LVE 2RHE1QAC 2Q1E 1IVL

L5* − L5* + 3T0W L5* + 3T0W

PDB ID Figure 3 ID Name Light chain Pure rotation (°) Orientation X Rotation (°) Y Rotation (°) Z Rotation (°)

1LVE E LEN κ 0.0 scFv L5* - L5* λ 89.5 49.3 29.1 -60.5 L5* + L5* (MG) λ 102.1 32.6 29.9 -86.1

3T0W M8 M8 λ 130.9 105.8 51.5 -34.7 2RHE H RHE λ 61.0 -59.0 -6.6 -11.6 2Q1E G AL-09 + 7 mut. κ 88.9 87.2 8.9 -10.7 1IVL I M29B κ 174.1 186.0 0.1 -11.2 1EK3 REC κ 177.7 Flipped 177.5 31.5 -0.3

2KQM KI 018/08 Y87H κ 171.9 Flipped 169.0 34.6 -7.9 1QAC F LEN Q89L κ 172.3 Flipped 170.8 28.1 -4.7 3LVE LEN Q38E κ 179.7 Flipped 179.8 28.6 0.2 4LVE LEN K30T κ 180.0 Flipped 180 24.4 -0.1 5LVE LEN Q89A κ 173.6 Flipped 187.0 25.4 1.8

(a)

(b) * 20 * 40 * 60 * 80 * 100 * L5* : QAVVTQEP-SVTVSPGGTVILTCGSSTGAVTSG--HYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSLSDVD---GYLFGGGTQLTVLS-- : 1103T0W : XP.L..S.-..SGT..QK.TIF.SG.SSN.ED---NSVY.Y..F..TT.KV..YND.RRS.GV.D.....KS.TS.S.A..GLRS....D...LSW.DS-LN.WV.....KV...D-- : 1112FB4 : .S.L..P.-.ASGT..QR.TIS.SGTSSNIG.---STV..Y..L..M..KL..YRDAMRP.GV.D.....KS..S.S.A.GGL.S...TD...AAW..S-LNA.V..T..KV...GQP : 1132FL5 : SYELK.P.-..S....Q.ARI..S---.D.LPK--K..Y.Y.ERS....VLVVY.DSGRP.EI.E.....SS.T..T....G..V....D...YSDISN--GYP......K.S.GQPK : 1102IG2 : .S.L..P.-.ASGT..QR.TIS.SGTSSNIG.---STV..Y..L..M..KL..YRDAMRP.GV.D.....KS..S.S.A.GGL.S...TD...AAW..S-LNA.V..T..KV...GQ- : 1123C2A : .S.L..P.-..SAA..QK.TIS.SG.SSNIGN---N.VL.Y..F..T..KL..YGNN.RP.GI.D.....KS.TS.T.G.TGL.TG...D.F.ATW.SGLSADWV.....K.....-- : 1123GBM : --.L..P.-..SAA..QK.TIS.SG.SSNIGN---D.VS.Y..L..T..KL..YDNN.RP.GI.D.....KS.TS.T.G.TGL.TG...N...ATW.R--TAYVV.....K..QPK-- : 1083GBN : --.L..P.-..SAA..QK.TIS.SG.SSNIGN---D.VS.Y..L..T..KL..YDNN.RP.GI.D.....KS.TS.T.G.TGL.TG...N...ATW.RRPTAYVV.....K....EE- : 1117FAB : AS.L..P.-..SGA..QR.TIS.TG.SSNIGA.--.NVK.Y..L..T..KL...HNN-------A...V.KS.TS.T.A.TGL.A....D...QSY.R---SLRV.....K....RQ- : 1048FAB : --EL..P.-..S....Q.ARI..S---ANALPN--Q..Y.Y.....R..VMV.YKDTQRP.GI.Q...S.TS.TTVT....GV.A....D...QAW.N---SASI.....K....G-- : 1051FLR : DV.M..T.L.LP..L.DQASIS.R..QSL.H.NGNT.LR.YL.....S.KV..YKVSNRF.GV.D.....GS.TDFT.K..RVEA..LGV.F..Q.THVP---WT.....K.EIKRA- : 114

CDR1 CDR2 CDR3

L5* 2FB4

CDR1

CDR2

CDR3

MG

V FRL

V FRH

Fig. S3. Quaternary structure comparison of VL/VL interfaces. (a) Survey of VL/VL dimers. 61 light chain dimers were found in the PDB (04/04/2011). Nine of these VL dimers form a non-scFv-like quaternary arrangement, and are listed in this table, as are L5* and M8 VL dimers. All of these dimer complexes except M8 (3T0W) possess C2 symmetry, but differ in the rotation angle and translational displacement between monomers. Figure 3 of the main text illustrates domain-domain contacts of several of these dimers as listed. 1LVE (LEN) forms a scFv-like dimer, and has been chosen as the reference structure because several LEN point mutants are non-scFv-like. The table lists results of a pairwise comparison between each dimer to 1LVE, implemented by superimposing the A chains, and then calculating how many degrees of rotation are required to superpose the B chains. 1LVE is positioned with its VL/VL C2 symmetry axis along Y, and the ‘elbow’ angle along X. Results are given as the pure rotation angle, and also as 3 clockwise rotations around fixed X, Y and Z axes. The ‘flipped’ arrangement of VL/VL dimer has been previously described in the literature [4]. Ribbon cartoons with CDRs are shown for those complexes presented in Figure 3. Inset shows L5* and M8 VL domains with their respective ligands MG (yellow) and dimethylindole red, which binds as two crystallographic conformers (yellow and red). Arrows show the locations of the binding cavity openings. (b) L5* VL framework residues that contact CDRs of partner VL differ from conserved λ VL residues that mediate canonical VL/VH framework/framework contacts. L5* VL is shown aligned to M8 (3T0W) and 4-4-20 (1FLR), and to all human λ VL domains available as VL/VH structures in the PDB. VL framework residues that make major contacts (>10 Å2) with VH framework residues are highlighted in pink; VL framework residues that make major contacts to CDR residues (mostly CDR3) in partner VL are highlighted in blue. In depicted structures, these VL residues are shown as beige surfaces; in 2FB4, VH residues that make framework contacts with VL are shown as a gray surface.

S7

Chain A Chain B Contact (Å2)

TYR 32A TYR 55B 26.2

TYR 32A SER 56B 10.2

TYR 32A TRP 57B 25.6

PHE 36A TYR 96B 42.6

GLN 42A GLN 1B 2.2

ALA 43A GLN 1B 22.1

ALA 43A ASP 94B 9.2

ALA 43A LEU 97B 31.4

PRO 44A GLY 95B 2.3

PRO 44A TYR 96B 51.7

ARG 45A VAL 93B 28.2

ARG 45A ASP 94B 30.6

ARG 45A GLY 95B 9.8

ARG 45A TYR 96B 2.9

ALA 46A SER 91B 19.8

ALA 46A ASP 92B 2

ALA 46A TYR 96B 9.2

PHE 49A PHE 49B 5.8

PHE 49A GLU 50B 25.2

GLU 50A PHE 49B 30.3

TYR 55A TYR 32B 25.2

TYR 55A GLU 50B 4.1

TYR 55A SER 91B 3.7

TYR 55A TYR 96B 2.3

TRP 57A 11.7

TRP 57A TYR 32B 17

TRP 57A SER 91B 10.5

TRP 57A ASP 92B 36.3

TRP 57A VAL 93B 43.7

SER 91A ALA 46B 16.1

SER 91A TYR 55B 2.9

SER 91A TRP 57B 8.1

ASP 92A ARG 45B 3.7

ASP 92A TRP 57B 23.8

VAL 93A ARG 45B 23.7

VAL 93A TRP 57B 45.7

ASP 94A ALA 43B 6.4

ASP 94A ARG 45B 30.3

GLY 95A ALA 43B 6.1

GLY 95A PRO 44B 13.2

GLY 95A ARG 45B 8.9

TYR 96A PHE 36B 35.2

TYR 96A PRO 44B 32.6

TYR 96A ALA 46B 15.5

TYR 96A TYR 55B 2

TYR 96A TYR 96B 7.3

LEU 97A ALA 43B 23.8

LEU 97A PRO 44B 3.6

PHE 98A PHE 98B 25.1

Chain A Chain B Contact (Å2)

TYR 32A 23.1

ASN 34A 20.6

PHE 36A 45.1

ALA 46A 5.4

TYR 55A 42.2

SER 89A 46.4

LEU 90A 3.4

SER 91A 35.3

TYR 96A 48.5

PHE 98A 22.8

TYR 32B 14.1

ASN 34B 27.6

PHE 36B 41.1

ALA 46B 5.8

TYR 55B 44.2

SER 89B 49.4

SER 91B 41.6

TYR 96B 39

PHE 98B 20.1

CDR1

CDR2

CDR3

L5* (-MG)VL/V contactsL

L5* (+ MG)VL/V contactsL

L5* (+ MG)VL /MG contacts

876 Å2 964 Å2 576 Å2

Chain A Chain B Contact (Å2)

GLY 30A SER 56B 2.9

GLY 30A TRP 57B 11.6

TYR 32A PHE 49B 8.7

TYR 32A LYS 54B 3.2

TYR 32A TYR 55B 42

TYR 32A SER 56B 34.7

TYR 32A TRP 57B 17.9

ASN 34A ASN 34B 6.9

ASN 34A TYR 55B 8.4

PHE 36A TYR 96B 30.1

ALA 43A ASP 94B 19.7

PRO 44A GLY 95B 18.9

PRO 44A TYR 96B 42.2

ARG 45A VAL 93B 22

ARG 45A ASP 94B 31.8

ARG 45A GLY 95B 22.6

ARG 45A TYR 96B 4.1

ALA 46A SER 91B 6.5

ALA 46A GLY 95B 2.2

ALA 46A TYR 96B 12.2

PHE 49A TYR 32B 3.1

PHE 49A PHE 49B 41.5

PHE 49A GLU 50B 32.8

GLU 50A PHE 49B 29.5

LYS 54A TYR 32B 4.8

TYR 55A TYR 32B 49

TYR 55A ASN 34B 8

TYR 55A SER 91B 2.7

SER 56A GLY 30B 2.6

SER 56A TYR 32B 20

TRP 57A GLY 30B 17.3

TRP 57A TYR 32B 21.3

TRP 57A SER 91B 29.1

TRP 57A ASP 92B 29.9

TRP 57A VAL 93B 44.6

SER 91A ALA 46B 6.6

SER 91A TRP 57B 28.1

ASP 92A TRP 57B 26.6

VAL 93A ARG 45B 32.6

VAL 93A TRP 57B 32.8

ASP 94A ALA 43B 16.8

ASP 94A ARG 45B 4.9

GLY 95A ALA 43B 2.9

GLY 95A PRO 44B 18.4

GLY 95A ARG 45B 7.9

TYR 96A PHE 36B 32.3

TYR 96A PRO 44B 46

TYR 96A ALA 46B 13.4

TYR 96A PHE 98B 5.2

TYR 96B 4.9PHE 98A

GLY 30B

Fig. S4. Contact analysis of unliganded and liganded L5* VL/VL complexes. Surface contacts were calculated on the SPACE server [5] using default settings with 4.0 Å distance and 2.0 Å

2 surface area cut-offs. Outlined boxes identify amino acids that

make simultaneous contact to MG and the partner VL.

S8

Fig. S5. Features of L5* binding region. (a) Symmetry and packing of CDRs. Shown are 1.4 Å solvent accessible surface (SAS) renderings; small ribbon cartoon shows general location of CDRs relative to full dimer. More extensive inter-domain contacts exist for the liganded complex, especially between CDR1 and CDR2, as can be seen in the back view. (b) Analysis of binding cavities. SAS depictions of top and side views of cavities are respectively oriented looking along C2 symmetry axis (arrows) into cavity. Tiny red spheres represent allowable centroid positions of water molecules (1.4 Å probe) whose combined outer surface defines the cavity boundaries (using Hollow software) [6]. The discontinuous distribution of centroid positions defines sub-compartments between which water-sized molecules cannot exchange without protein dynamics. (c) Exposure of Ser89 enables contact with MG. Phenyl ring edge is inserted into cleft that opens upon disruption of Tyr96-Ser89 and Ser89-Asn34 hydrogen bonds. In this SAS depiction, black mesh highlights side chains of all residues that make significant contact to MG.

CDR1

CDR2

CDR3

MG

H O2

FR A

Y96

F98

S91

Y32

N34

Y55F36

unliganded

Y96

F98

S89 S91

Y32

N34

Y55F36

liganded

(c)

topside

(b)

CDR1

CDR2

CDR3

MG

H O2

liganded unliganded

front

back

(a)

S9

Fig. S6. Conformation of MG. (a) Electron density map of MG bound to AB dimer in the L5* crystal cell. (b) Comparison of conformers of L5*-bound MG and free MG. Left, front view along C2 axis of the L5* AB dimer (yellow) and the energy minimized structure of free MG cation (purple) (PubChem 3D CID 1129581), superposed via Ring 1. Right, front view along C2 axis of these ligands, superposed via the triarylmethane plane defined by carbon atoms 2, 8 and 14, as described below. (c) Conformations of MG in L5* crystal cell. Left, numbering of malachite green atoms. Molecules were built with partial double bonds; bond lengths from C1 to C8 and C1 to C14 are 1.44 ± 0.005 Å, and 1.48 ± 0.004 Å from C1 to C2. Angle measurements are made between a ring plane and the plane defined by central atoms C2, C8 and C14. Middle, tabulation of propeller angles in crystal cell. Right, largest difference in ring angles of MG bound to L5*. Shown are MG molecules I and L that have an approximate 12° difference in ring angle for Ring 2 (C8-C13). The differences (shown in Å) in atomic position for atoms in that ring are nonetheless small. These rotation angles differ markedly from those calculated for the minimum energy conformation of free MG cation, one of two equivalent resonance structures with a double bond (90º for R1 and R2, 0º for R3) [7].

(a) (b)

R1

R2

R3

R1

R2

R3

GH/L

AB/I

Dimer/MG R1 R2 R3

AB/I 40.4º 39.2º 31.5º

CD/J 41.2º 30.2º 30.6º

EF/K 38.3º 32.5º 26.1º

GH/L 42.8º 27.1º 33.0º

(c)

S10

FAP Count-rate (kHz)

Diffusion(µs)

Counts/Molecule

Number/Volume

Radius (nm)

Cy5 51 213 14.9 2.1 0.61

L5*(monomer)

67 475 13 5.1 1.4

dL5*(dimer)

52 464 12.7 4.1 1.3

dL5** (dimer)

46 487 10.2 4.5 1.4

(b)

dL5* Covalent Dimer 1230 bp

AGA2

Original L5*

Codon Optimized L5* 4x (GGGGS) linker

3x (GGGGS) linker

HA tag c-myc tag

d

m

(a)

CAGGCCGTCGTTACCCAAGAACCTAGTGTTACCGTTAGCCCAGGTGGTACTGTTATACTTACTTGTGGAAGTTCTACGGGTGCCGTCACATCTGGTCATTATGCAAATTGGTTTCAACAAAAACCAGGACAAGCTCCAAGAGCTTTGATTTTTGAAACTGATAAGAAGTATTCTTGGACCCCAGGTAGATTTTCTGGATCTTTGCTGGGAGCAAAGGCAGCTTTGACAATATCAGATGCTCAGCCTGAGGACGAAGCCGAGTATTACTGTTCTCTTAGCGACGTGGATGGCTACTTGTTTGGCGGTGGAACACAACTGACGGTTCTGTCC CAGGCTGTGGTGACTCAGGAGCCGTCAGTGACTGTGTCCCCAGGAGGGACAGTCATTCTCACTTGTGGCTCCAGCACTGGAGCTGTCACCAGCGGTCATTATGCCAACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGGCACTTATATTCGAAACCGACAAGAAATATTCCTGGACCCCTGGCCGATTCTCAGGCTCCCTCCTTGGGGCCAAGGCTGCCCTGACCATCTCGGATGCGCAGCCTGAAGATGAGGCTGAGTATTACTGTTCGCTCTCCGACGTAGACGGTTATCTGTTCGGAGGAGGCACCCAGCTGACCGTCCTCTCC

GGTGGTGGCGGCTCTGGTGGCGGTGGCAGCGGCGGTGGTGGTTCCGGAGGCGGCGGTTCT

EAEAYGGGGSGGGGSGGGGSGGGGS

STGHHHHHH

QAVVTQEPSVTVSPGGTVILTCGSSTGAVTSGHYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSLSDVDGYLFGGGTQLTVLS QAVVTQEPSVTVSPGGTVILTCGSSTGAVTSGHYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSLSDVDGYLFGGGTQLTVL

Fig. S7. Spectral evidence that L5* ternary complex is the fluorescent form. (a) Design of covalently linked dL5* dimer as displayed on the yeast cell surface. Construct encoding protein (red letters) secreted from yeast lacks the upstream HA tag and adjacent 3x (GGGGS) linker (black letters), and replaces the c-myc tag with a 6-his tag used for protein purification. SDS PAGE gel shows purified dimer (d) and monomer (m) protein. (b) Fluorescence correlation spectroscopy. 10 µM protein was equilibrated with 4 nM MG-2p in PBS+ buffer. Cy5 was used as a calibrant. For reference, GFP has a hydrodynamic radius of 1.6 nm.

S11

ΔH (kJ/mol)

ΔG = -60

ΔG = 0

-TS

(kJ

/mol)

Δ

-120 -100 -80 -60 -40 -20 0 20 40

-60

-40

-20

0

20

40

60

80

100

Enthalpy driven

Entropy driven

dL5*

Biological

Miscellaneous

Synthetic

Ligands

Fig. S8. Thermodynamic parameters of binding MG to the linked dimer dL5* compared to other protein/small molecule complexes. Graph presentation follows that of Ladbury and colleagues [8], and plots all isothermal titration calorimetry (ITC) information in the SCORPIO 2 database as of 9/21/12. For the binding of MG to dL5* ( ), the ΔG value (-59 kJ mol

-1) is

calculated from separate equilibrium binding measurements (KD = 90 pM), and the -TΔS value (8 kJ mol-1

) is derived from the ΔG value and the ITC experimental value of ΔH (-67 kJ mol

-1) (see Fig. 8 in main text). With the singular exception of

streptavidin/biotin ( ), the ΔH contribution to dL5*/MG formation is notably greater than for other studied high affinity protein/ligand complexes (ΔG < 50 kJ/mol), such as gyrase B bound to Novobiocin ( ) and HIV protease bound to several structurally unrelated inhibitors ( ).

S12

Distance (m)

16

-bit

Gra

ysca

le

0 20 40 600

20000

40000

60000

01

,00

02

,00

0

110

210

310

410

510

11,268

Cell

Count

Fluorescence (635 nm ex. 685/30 nm em.)

100 nM MG

DIC

Op

tima

lL

ine

ar

Distance (m)

16

-bit

Gra

ysca

le

0 20 40 600

2000

4000

6000

02

,00

01

,00

0

2102-10 410 5103100

821

Cell

Count

Fluorescence (635 nm ex. 685/30 nm em.)

100 pM MG

DIC

Op

tima

lL

ine

ar

Fig. S9. Improved in vivo visualization and cytometry using dL5**. Yeast cells display on their surface genetically fused dimer (dL5**) encoded as a single copy chromosomal integrant. Confocal microscope images, image transect profile, and flow cytometry histogram represent cells in PBS+ containing 100 pM MG-2p (left) or 100 nM MG-2p (right). Laser confocal imaging was carried out on a Zeiss LSM510 using identical instrument settings with 633 nm laser excitation and 650-710 nm bandpass emission. Linear micrographs represent unadjusted 16-bit data that has been inverted to show as grayscale; optimal micrographs have been contrast-adjusted using ‘best-fit’ setting on Zeiss software prior to inversion. Scan profiles were carried out on linear micrographs; the transects are shown as red dotted lines on best-fit micrographs to facilitate visualization. Fluorescence distribution in a population of 100,000 cells were determined on a FACS-Diva flow cytometer using 635 nm laser excitation with a 670-700 nm bandpass emission filter. Mean fluorescence values of 821 (100 pM) and 11,268 (100 nM) are large compared to control values of about 7 (no dye) and 9 (100 nM) for cells not expressing FAP (data not shown).

[1] Szent-Gyorgyi C, Schmidt BF, Creeger Y, Fisher GW, Zakel, KL, Adler, S, et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat Biotechnol 2008;26:235-40.

[2] Szent-Gyorgyi C, Schmidt BF, Fitzpatrick JAJ, Bruchez MP. Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes. J Am Chem Soc 2010;132:11103-09.

[3] Abhinandan, KR, Martin, AC. Analyzing the "degree of humanness" of antibody sequences. J Mol Biol 2007;369:852-62.

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