Biomembrane Electrochemistry - ACS Publications - American

15

Downloaded by UNIV OF BATH on July 1, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch015

Design Principles and Chemical Synthesis of Oligomeric Channel Proteins John M . T o m i c h , Anne Grove , Takeo Iwamoto , Stephan M a r r e r Myrta S. Montal , and Mauricio M o n t a l 1,3

2

2

1,3

2,4

,

2,5

Department of Biochemistry, University of Southern California Medical School and Children’s Hospital, Los Angeles, C A 90054-0700 Department of Biology, University of California, San Diego, L a Jolla, C A 92093-0319 1

2

Proteins that emulate pore properties of the dihydropyridine-sensitive calcium channel or the nicotinic acetylcholine receptor have been designed and synthesized. The designed proteins consist of bundles of amphipathic α-helices with sequences that correspond to specific segments of authentic proteins and are arranged such that charged or polar residues line an aqueous pore. Molecular models suggest that such structures satisfy geometric and functional requirements to con stitute the pore-forming element of channel proteins. The designed proteins are synthesized by solid-phase methods and purified. The single-channel conductance properties of designed proteins are studied in lipid bilayers. The synthetic proteins mimic the ionic conductance, selectivity, and pharmacological properties of authentic channel pro teins. Synthetic proteins that represent segments of the authentic proteins that are not predicted to line an aqueous pore do not mimic the targeted biological activity.

3

4

Current address: Department of Biochemistry, Kansas State University, Manhattan, KS 66506-3702 Current address: F. Hoffman-LaRoche Ltd., PSQA-2, Ch-4002 Basel, Switzerland Corresponding author

0065-2393/94/0235-0329$09.08/0 © 1994 American Chemical Society

Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

330

Ν

BIOMEMBRANE ELECTROCHEMISTRY

E U R O T R A N S M I T T E R R E C E P T O R S A N D IONIC C H A N N E L S are

responsible

for

two fundamental properties o f the b r a i n : electrical excitability a n d synaptic transmission. N e u r o n a l signaling occurs through synaptic junctions between the nerve endings a n d the b o d y o f the nerve c e l l or other effector cells. C o n d u c t i o n a n d propagation o f nerve signals through nerve cells a n d axons are mediated b y voltage-gated channels ( J). I n neurons, as i n other excitable cells, the influx o f C a v i a voltage-sensitive c a l c i u m channels generates b o t h electrical a n d c h e m i c a l signals. C a l c i u m influx carries depolarizing charge that contributes to electrical activity a n d also leads to a rise i n intracellular c a l c i u m concentration f r o m extracellular fluid o r b y release f r o m intracellular stores. Changes i n c a l c i u m concentration constitute a c h e m i c a l message for calcium-sensitive mechanisms that control, for example, i o n channel gating a n d transmitter release ( I , 2).


2 +

Transfer o f i n c o m i n g signals at the c h e m i c a l synapses present at nerve terminals occurs by releasing neurotransmitters f r o m their presynaptic t e r m i nal a n d effecting a change i n i o n permeability o f the postsynaptic cells. T h e channels i n the postsynaptic m e m b r a n e are often chemically gated; that is, they open or close i n response to transmitter b i n d i n g . Acetylcholine ( A C h ) is released by the presynaptic n e u r o n . T h e b i n d i n g o f A C h to A C h receptors f o u n d o n postsynaptic membranes o f nerve terminals a n d neuromuscular endplates results i n the transient o p e n i n g o f cation-selective channels that are responsible for depolarizing the postsynaptic c e l l ( I ) . P r e - a n d postsynaptic elements, therefore, play a key role i n excitation o f the b r a i n . W e focus here o n prototypes o f a presynaptic a n d a postsynaptic element. A s an example o f a presynaptic element, w e selected the dihydropyridine-sensitive c a l c i u m channel, a m e m b e r o f the superfamily o f voltage-gated channel proteins. T h e most extensively studied m e m b r a n e protein, the nico tinic cholinergic receptor, w h i c h belongs to the family o f ligand-gated chan nels, is present i n the postsynaptic m e m b r a n e . These two examples are used to describe a strategy that aims to identify sequence-specific motifs that are responsible for the performance o f u n i q u e functions a n d to outline an experimental approach to evaluate identified structural motifs.

Inferences about Channel Protein Structure K e y functional elements o f voltage- a n d ligand-gated ionic channels are the mechanisms b y w h i c h their conformation changes i n response to ligand b i n d i n g or variations i n m e m b r a n e electric field; the sensor that detects a stimulus and couples it to the o p e n i n g a n d closing o f the channel; the permeation pathway, specifically pore size a n d ionic selectivity o f the o p e n channel; a n d the sites o f action o f drugs a n d toxins that specifically m o d i f y properties o f the channel (J). M o l e c u l a r c l o n i n g a n d sequencing l e d to the elucidation o f p r i m a r y structures o f several superfamilies of voltage- and


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Oligomenc Channel Proteins


ligand-gated channels ( 3 ) . H o w e v e r , no high-resolution structural information is yet available. T h e challenge is, therefore, to try to identify f r o m the p r i m a r y sequences the structural elements that may determine the functional proper ties o f channel proteins (4, 5 ) . A search for sequences that are compatible w i t h a given function, such as the p o r e - f o r m i n g structure o f channel proteins, originated w i t h the wealth o f sequence information available o n voltage- a n d ligand-gated channel proteins (3). Extensive sequence homology a m o n g members o f these families o f proteins is evident; characteristic structural features i n c l u d e the occurrence o f homologous subunits oriented across the m e m b r a n e a n d organized as a symmetric o r pseudosymmetric array a r o u n d a central aqueous channel a n d the presence o f segments capable o f adopting α-helical transmembrane structures. It is plausible, therefore, that a unifying structural m o t i f i n the biological design o f i o n channels is a cluster o f amphipathic α-helices arranged such that charged o r polar residues line the central h y d r o p h i l i c pore a n d nonpolar residues face the h y d r o p h o b i c environment o f the p r o t e i n a n d the bilayer interior (4, 5 ) . S u c h a structure m a y account f o r geometric requirements, determine ionic specificity, a n d explain the diversity o f channel proteins based o n sequence specificity a n d oligomer size.

Identification of Possible Pore-Lining Segments Identification o f p r e s u m e d p o r e - l i n i n g segments is based o n the p r i m a r y structure o f the channel p r o t e i n a n d knowledge o f the physiology o f the molecule. A m p h i p a t h i c α-helical segments greater than 20 residues i n length — s u f f i c i e n t to span the w i d t h o f the bilayer c o r e — a r e identified b y the use of e m p i r i c a l secondary structure predictors; specific residues allow for appro priate ionic selectivity (6-8). T h e d i h y d r o p y r i d i n e - ( D H P ) - s e n s i t i v e c a l c i u m channel p u r i f i e d f r o m skeletal muscle is c o m p o s e d o f five subunits: α a , β, y, a n d δ ( 9 - 1 2 ) . T h e α subunit (molecular weight M ~ 170 k D ) forms a functional voltage-gated calcium channel ( 9 , 13-15) a n d contains the b i n d i n g sites for the three classes o f c a l c i u m channel modulators: 1,4-dihydropyridines (16), phenylalkylamines, a n d benzothiazepines (17). T h e p r i m a r y structure o f the a subunit was first elucidated f r o m skeletal muscle (18); highly homologous sequences have since b e e n c l o n e d f r o m cardiac muscle ( 1 3 , 19, 20) a n d b r a i n (21), aorta (22), and l u n g (23) tissue. 1 ?

2

2

r

1

Extensive sequence homology is evident between c a l c i u m (13, 18-23), sodium (24-29), a n d potassium channel proteins (30-34). T h e primary structure o f s o d i u m a n d c a l c i u m channel proteins suggests the occurrence o f four homologous domains w i t h the four repeats organized as pseudosubunits around a central pore (4, 24), whereas potassium channels are considered to be constructed o f a cluster o f four subunits ( 3 5 , 36), each o f w h i c h corre-


332


sponds to a single repeat o f s o d i u m o r c a l c i u m channels. E a c h repeat contains six segments ( S 1 - S 6 ) that are p r e d i c t e d to f o r m α-helical transmembrane structures. A c o m m o n f u n c t i o n o f S3 segments f r o m c a l c i u m , s o d i u m , a n d potassium channel proteins is suggested b y the extensive sequence homology, particu larly w i t h respect to the position o f negatively charged o r polar residues that may b e i n v o l v e d i n l i n i n g a cation-selective channel. F o u r S3 segments m a y f o r m a b u n d l e o f α-helices that create the transmembrane pore (4, 5, 37-39). T h e A C h receptor ( A C h R ) f r o m Torpedo californica ( M « 250 k D ) is c o m p o s e d o f four glycoprotein subunits w i t h a stoichiometry o f α β 7 δ that are assembled as a pentamer w i t h the i o n channel i n the center (40-42). G a t i n g o f currents through these channels depends o n b i n d i n g o f ligands such as A C h to specific sites o n the α subunits. Analysis o f the p r i m a r y structure l e d to the assignment i n each subunit o f four h y d r o p h o b i c trans m e m b r a n e segments, M 1 - M 4 ( 3 , 42). T h e A C h R c h a n n e l is an aqueous pore that selects against anions b u t passes cations a n d m a n y nonelectrolytes < 7 Â diameter ( I ) . Theoretical a n d permeation studies suggest that the vestibules contain a net negative charge that attracts cations into the pore, w h i c h is p r e s u m e d to b e fined w i t h polar uncharged residues (43). L a b e l i n g o f the channel b y noncompetitive inhibitors such as chlorpromazine (44) a n d t r i p h e n y l m e t h y l p h o s p h o n i u m (45, 46) has identified serine-262 i n M 2 o f the δ subunit as part o f a high-affinity site. H o m o l o g o u s regions f r o m the different subunits contribute to the u n i q u e high-affinity site (47). Consequently, the M 2 segment o f the δ subunit is thought to be i n v o l v e d i n f o r m i n g the A C h R channel ( 5 , 48-51). r


2

Design of Pore-Forming Proteins T o demonstrate the existence o f functional elements responsible f o r pore properties o f channel proteins, peptides w i t h sequences that represent such functional segments are synthesized a n d their ability to m i m i c the targeted biological activity is tested b y incorporation o f the peptides into l i p i d bilayers. T h i s approach allows r a p i d determination o f w h i c h p r e s u m e d transmembrane helices m a y f o r m functional channels. T h e peptides self-assemble i n the m e m b r a n e to generate conductive oligomers, presumably w i t h h y d r o p h o b i c surfaces that face the p h o s p h o l i p i d a n d h y d r o p h i l i c residues that fine the pore. Channels o f different sizes (oligomerie n u m b e r ) result (37, 48). T h e occurrence o f a cluster o f amphipathic α-helical peptides that forms the i n n e r b u n d l e o f c h a n n e l proteins a n d the determination o f oligomerie n u m b e r m a y b e addressed t h r o u g h the design a n d synthesis o f larger polypeptides that w i l l pack i n a predictable m a n n e r to y i e l d a p r o t e i n w i t h p r e d e t e r m i n e d conformational properties. Considerable effort has b e e n de-


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voted to the design a n d c h e m i c a l synthesis o f large polypeptides that assume tertiary structures. These approaches i n c l u d e de novo design o f a four-helix b u n d l e p r o t e i n ( 5 2 ) a n d a m i m i c o f the serine protease catalytic t r i a d ( 5 3 ) . A p r o m i s i n g strategy f o r the construction o f proteins w i t h

predetermined

structure involves the covalent attachment o f peptide segments w i t h a h i g h potential for secondary structure to a m u l t i f u n c t i o n a l carrier molecule (tem plate) to generate nonlinear p r o t e i n molecules that exhibit t e m p l a t e - i n d u c e d secondary structure; the product o f attachment is the template-assembled synthetic p r o t e i n ( T A S P ) (54-56).

A linear assembly o f peptide blocks

necessitates a specific f o l d i n g process to achieve the p r o p o s e d conformation, whereas the template molecules direct the attached peptide segments into


the r e q u i r e d conformation. T h e template molecule m a y b e any m u l t i f u n c tional molecule w i t h p r o p e r spatial arrangement o f attachment

sites a n d

l i m i t e d conformational flexibility. F o r example, spatial constraints i n d u c e d b y attaching a m p h i p h i l i e α-helix-forming peptides to a template enhance p e p tide interactions a n d act as the major driving force to f o l d the templateassembled peptides into the p r e d e t e r m i n e d conformation

(57).

Proteins that m i m i c the p r e s u m e d p o r e - f o r m i n g structure o f the D H P sensitive c a l c i u m channel a n d the nicotinic A C h R were designed using the TASP (54-56)

strategy.

A nine amino acid template

molecule

(KKKPGKEKG)

that allows f o r attachment o f peptides at the e-amino groups o f four

lysines was used to generate four-helix b u n d l e structures (49) b y the assem b l y o f four identical peptides. ( N o t e that the standard one-letter amino acid code is used.) E a c h peptide represents the sequence o f the S3 segment f r o m the fourth internal repeat o f the D H P - s e n s i t i v e c a l c i u m channel ( T C a I V S 3 ) 4

(13, 18, 21) o r the Μ 2 δ segment o f the nicotinic A C h R ( Τ Μ 2 δ ; F i g u r e 1A) 4

( 5 8 ) . T h i s approach has generated a novel class o f m e m b r a n e associating, p o r e - f o r m i n g molecules (synporins) (49). T h e devised nomenclature specifies a template w i t h four attachment

sites ( T ) , f o l l o w e d b y the conventional

designation o f i n d i v i d u a l transmembrane

4

segments.

A homotetramer o f I V S 3 ( T C a I V S 3 ) is a plausible m o d e l o f the p r o 4

posed heterotetramer that forms the pore o f the authentic D H P - s e n s i t i v e calcium channel. T h e sequence o f I V S 3 is conserved between skeletal muscle and isoforms o f cardiac muscle, brain, a n d aorta, a n d e m p i r i c a l secondary structure predictors (6-8)

suggest that the peptide forms an amphipathic

α-helix (hydrophobic m o m e n t = 0.19) w i t h a length sufficient to traverse the hydrocarbon core o f the m e m b r a n e . S u c h features are also characteristic o f the other three S3 segments because o f extensive sequence homology. F u r t h e r , a synthetic peptide w i t h the sequence o f a homologous S3 segment o f the b r a i n s o d i u m channel forms cation-selective channels i n l i p i d bilayers (37). M 2 exhibits h i g h homology b o t h between subunits o f the Torpedo α β7δ 2

AChR

complex a n d between species ( 3 ) . Secondary structure predictors

suggest that Μ 2 δ segments c o u l d f o r m amphipathic α-helices ( ( μ > = 0.25).


334


Similarly, a synthetic peptide w i t h a sequence that represents the Μ 2 δ segment o f the Torpedo A C h R forms discrete ionic channels i n l i p i d bilayers w i t h conductance properties comparable to those o f the authentic A C h R channel (48).


Four-Helix Bundles: A Plausible Model for the Pore Structure of Channel Proteins F o r the construction o f a four-helix b u n d l e , the spatial orientation o f template amino acids is considered. T h e conformational characteristics o f the side chains o f lysine predict a d i r e c t i o n p e r p e n d i c u l a r to the plane o f the t e m plate, a n d a l l attachment sites f o r peptide modules m a y b e o r i e n t e d cis relative to the plane o f the template backbone. E n e r g e t i c considerations c o n f i r m that a b u n d l e o f four α-helices is a reasonable m o d e l f o r the pore structure o f c h a n n e l proteins. M o d e l s w e r e generated using existent coordinates f o r the s o d i u m c h a n n e l S3 h o m o tetramer (39) b y specific residue replacements. T h e I N S I G H T a n d D I S C O V E R p r o g r a m packages ( B i o s y m Technologies, Inc., San D i e g o , C A ) w e r e used. O p t i m i z e d helical structures f o r the homotetramers o f T C a I V S 3 a n d Τ Μ 2 δ are shown i n F i g u r e I B a n d C . T h e template p o r t i o n o f the molecules is m o d e l e d as a β h a i r p i n w i t h attachment sites f o r the channelf o r m i n g peptides a l l cis to the plane o f the template ( F i g u r e I B ) . T h e attached α-helical modules are parallel a n d aligned w i t h the c o u p l i n g sites at 4

4

(A)

Template

KKKPGKEKG

IVS3

DPWNVFDFLIVIGSIIDVILSE

IVS5

YVALLIVMLFFIYAVIGMQMFGK

M26

EKMSTAISVLLAQAVFLLLTSQR

M16

LFYVINFITPCVLISFLASLAFY

Figure 1. (A) Amino acid sequences of template and oligopeptides used to generate the proteins studied, pore-forming molecules T CaTVS3 and Τ Μ2δ, and proteins designed using hydrophobic sequences, T CaTVS5 and Τ Μ1δ. IVS3 corresponds to amino acids 1180-1201, TVS5 corresponds to residues 1269-1291 (18), and Μ2δ and Μίδ correspond to amino acids 255-277 and 226-248, respectively (58). Peptides are attached to template lysines indicated with an asterisk (*). Standard one-letter amino acid code is used. 4

4

4

4


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TOMICH E T AL.

Oligomeric Channel Proteins

335


(Β)

Figure 1.—Continued. Computer-generated molecular model of synthetic pore proteins. (B) Energy-optimized parallel tetramers of T CaIVS3 (upper) and Τ Μ2δ (lower). Β is a side view with the nonapeptide template at the top and the Ν terminus at the bottom of the two α-helical bundles (Marrer, S.; Montai, M., unpublished). Residues are colored according to hydrophobicity: red, acidic residues; blue, basic residues; orange, serine and threonine; bnght yellow, methionine; yellow, tryptophane and phenylalanine; pink, asparagine and glutamine; white, glycine and proline; green, lipophilic residues; light blue, α-carbon backbone and its ribbon representation; magenta, solvent-accessible surface (dotted). Continued on next page. 4

4


336



(C)

Figure 1.—Continued. Computer-generated molecular model of synthetic pore proteins. (C) Energy-optimized parallel tetramers of T CaIVS3 (upper) and T M2 8 (lower). C is the end view with the Ν terminus in front (89). Residues are colored according to hydrophobicity; see previous page. 4

4


15.

TOMICH ET AL.

OUgomeric Channel Proteins

337

the €-amino groups o f template lysines p r o v i d i n g the spatial organization f o r the four-helix bundles. T h e orientation o f the template relative to the b u n d l e is dependent o n the conformational properties o f attached helices. T h e N - t e r m i n a l residues correspond to the untethered e n d o f the helical modules a n d are assigned to the intracellular face o f the m e m b r a n e ( J , 18). T h e length o f the bundles is sufficient to span the l i p i d bilayer (32 Â ) ( 3 9 ) . T h e bundles show a left-handed twist w i t h an interhelical angle o f « 15°. F i g u r e 1 C shows an e n d v i e w o f the bundles; the Ν terminus is i n front. H y d r o p h o b i c a n d hydrophiUc residues occur o n opposite faces o f the helical cylinders. T h e solvent-accessible surface (dotted) discloses the symmetric nature o f the T C a I V S 3 pore (ring o f overlapping surface density). T h e square vestibule at the entry o f the pore o f T C a I V S 3 is f o r m e d b y indole nitrogens o f tryptophanes ( W - 3 ) that hydrogen b o n d w i t h aspartates ( D - l ) . T h e cross section is 7.9 Â, w h i c h corresponds to a distance between solventaccessible surfaces o f 4.5 Â. T h e l u m e n o f the pore is l i n e d w i t h p o l a r - n e u tral residues a n d two sequential clusters o f acidic residues, D - 7 a n d D - l 7 . T h e narrowest section o f the pore occurs at D - 7 ; the distance between opposing carboxylates is 7.4 Â w i t h 4.2 Â d e l i m i t e d b y the boundaries o f solvent-accessible surfaces. T h i s cluster o f negatively charged residues m a y provide a high-affinity b i n d i n g site f o r permeant cations ( 5 9 , 60). 4


4

T h e l u m e n o f the Τ Μ 2 δ pore is l i n e d w i t h p o l a r - n e u t r a l residues. S-8 corresponds to serine-262 i n the actual p r o t e i n sequence a n d faces the l u m e n o f the pore. Serine-262 is considered exposed to the l u m e n o f the pore a n d may participate i n the c o n d u c t i o n o f ions through the A C h R channel ( 3 9 , 44-46, 49-51). T h e cross section is 9.5 Â. F - 1 6 , w h i c h corresponds to phenylalanine-270 i n the p r o t e i n sequence, is exposed to the l u m i n a l surface and forms the narrowest section o f the pore (8.3 Â). E - l , w h i c h corresponds 4

to glutamate-255 o f the δ subunit sequence, hydrogen-bonds w i t h K - 2 o f each helix, a n d generates a ring o f charged residues. E - l corresponds to the postulated intracellular e n d o f the receptor channel, considered to b e nega tively charged ( 3 9 , 42, 43, 49, 61). H e n c e , bundles o f four amphipathic α-helices fulfill the structural a n d energetic requirements f o r the i n n e r b u n d l e that forms the pore o f the D H P - s e n s i t i v e c a l c i u m channel a n d the nicotinic A C h R channel.

Synthesis and Purification Synthesis o f the four-helix bundles was accomplished b y a two-step procedure (outlined as a flow chart i n F i g u r e 2). Proteins w e r e synthesized b y solid phase methods i n accordance w i t h general principles (62-64) using a peptide synthesizer ( m o d e l 431, A p p l i e d Biosystems ( A B I ) , Foster C i t y , C A ) . L - e o n figuration amino acid derivatives were used ( T h e P e p t i d e Institute, Osaka, Japan). T h e acid-labile terf-butyloxycarbonyl- ( i - B o c ) - p r o t e c t i n g groups w e r e


338


(A)


(^-BO^BN-HC-C-CZJ4

resin )

1

Θ

"Ν

-C-CH-NH-Qt-BocJ)

t-Boc >HN-HC-C=0

©

1. t - B o c

deprotection

2 . DCC coup I i ng w i t h 0.25

eq.t-Boe-fIBOC-Lys

3 . c a p p i n g of r e m a i n i n g s i t e s (Β)

A c

® :

-HN-HC-C-

C-CH-NH-OCH-NH

II 0

NH

A c

-(faoc^)

|-HN-HC-C=0

assembly of

template

Figure 2. Flow chart of the protocol used to synthesize tetrameric bundles of defined amino acid sequence. (A) The highly substituted polystyrene resin, 0.75 mmol/g, that contains t-Boc glycine (Gj attached through a Ρ AM linker (open box). (B) The most accessible sites are selected by coupling with only 0.25 equivalents of the t-Boc-fmoc lysine. The remaining unreacted sites are blocked using an excess of acetic anhydride (Ac).


15.

TOMICH E T AL.

339


(C)

E(oBzl) G

K(CI-Z)

ΛΛΑΛ ΛΑΑΛ/Ξ^ (


K(fmoc)

K(faôc)

1. t - B o c (D)

Κ (fiâoc)

deprotection

2. cappi ng

IKC1-Z)

p i p e r i d i n e d e p r o t e c t i o n of fmoc (Ε)

CC/HOBt coup 1i ng w i t h Boc Amino A c i d

Figure 2. —Continued. Flow chart of the protocol used to synthesize tetrameric bundles of defined amino acid sequence. (C) The completed template KKKPGKEKG. (D) The free Ν terminus of the template is blocked with excess acetic anhydride. (E) The fmoc groups are removed by treatment with 20% piperidine in DMF. Continued on next page.


340



Ι assembly of h e l i c a l

chains

DCC/HOBt coup I i ng

I

Figure 2. —Continued. Flow chart of the protocol used to synthesize tetrameric bundles of defined amino acid sequence. (F), (G) Single amino acids are added to sequentially assemble the peptide modules of the synthetic proteins. Continued on next page.

used f o r N protection. A l l c o u p l i n g steps were p e r f o r m e d i n N - m e t h y l p y r rolidone ( N M P ) containing d i m e t h y l sulfoxide ( D M S O ) f o r at least 30 m i n using a 10-fold excess o f p r e f o r m e d 1-hydroxybenzotriazole ( H O B T ) esters. C o u p l i n g efficiencies at each step were m o n i t o r e d b y the quantitative ninhyd r i n test (65). a



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341


2.wash w i t h e t h e r

Figure 2. —Continued. Flow chart of the protocol used to synthesize tetrameric bundles of defined amino acid sequence. (H) Single amino acid added to sequentially assemble the peptide modules of the synthetic proteins. Continued on next page.

Steric constraints o f the four-helix b u n d l e proteins require the use o f low-substitution resins for o p t i m a l synthetic y i e l d . R e d u c e d loading capacity o f the preloaded f - B o e - g l y e i n e - P A M (4-oxymethylphenylacetamidomethyl) resin ( 0 . 7 6 - m m o l / g capacity; A B I ) was accomplished d u r i n g c o u p l i n g o f the first amino acid: a r e d u c e d amount o f N - £ - B o c - N - f m o c a

(9-fluorenylmeth-

e

oxycarbonyDlysine (0.25 equivalent) was allowed to react w i t h the resin for 1 h ( F i g u r e 2 A ) . T h e remaining sites were c a p p e d w i t h acetic anhydride. T h i s strategy was designed to allow c o u p l i n g to only the most accessible sites o n the resin. B y use o f this lower substitution ( 0 . 1 - 0 . 3 m m o l / g ) , the n u m b e r o f recouplings r e q u i r e d to attain

> 9 9 . 5 % c o u p l i n g efficiencies was r e d u c e d

a n d increased weight gains were obtained. A

common

nine

amino

acid

template

Ac-K(N -fmoc)~K(N -Cle

e

Z)-K(N -fmoc)-P-G-K(N -fmoc)-E(7-OBzl)-K(N -fmoc)-G-PAM e

e

e

resin

(where A c is acetyl, C l - Z is 2-chlorobenzyloxycarbonyl, a n d O B z l is b e n z y l ester) was synthesized. F a i l e d sequences d u r i n g synthesis o f the

template

were c a p p e d using acetic anhydride ( F i g u r e 2 B and C ) . T h e sequence was c o n f i r m e d by automated E d m a n degradation o n a peptide sequencer ( A B I


342



(I)

M u l t i p l e HPLC pur i f i c a t i o n s

Figure 2. —Continued. Flow chart of the protocol used to synthesize tetrameric bundles of defined amino acid sequence, (i) HF cleavage releases the synthetic protein from the support and deprotects the amino acid sidechains. (]) Chromatography using HPLC generates the purified channel proteins. m o d e l 477A), a n d the N - t e r m i n a l lysine was b l o c k e d b y treatment w i t h 1 7 % acetic anhydride a n d 7 % diisopropylethylamine i n Ν M P for 10 m i n . A s s e m b l y o f peptide blocks typically starts w i t h 0.05 m o l o f template resin. T h e base-labile N - f m o c - p r o t e c t i n g groups were r e m o v e d b y i n c u b a tion w i t h 2 0 % p i p e r i d i n e (v/v) i n dimethylformamide for 20 m i n ( F i g u r e 2 D ) . T h e four peptide blocks were assembled simultaneously ( F i g u r e 2 E - G ) e


15.

TOMICH ET AL.

343


b y stepwise synthesis using H O B t - a c t i v a t e d esters o f N - £ - B o c amino a c i d a

derivatives o n a synthesizer ( A B I m o d e l 431). T h e side chains o f trifunctional amino acids were protected as follows: E ( O B z l ) , D ( O B z l ) or D(cyclohexyl ester) ( O c - H e x ) , S(benzyl ether) ( B z l ) , T ( B z l ) , R(tosyl), a n d W ( C H O ) . M u l t i ple couplings ( 3 - 5 ciencies

p e r residue) were p e r f o r m e d to ensure c o u p l i n g effi

> 9 9 . 5 % for each step. C a p p i n g o f failed sequences w i t h acetic

anhydride was i n c l u d e d i n some

syntheses.

Tracer

amounts

of [ H ]

t-

3

B o c - l e u c i n e were a d d e d to quantitate concentrations. Ν t e r m i n i were not capped. W e i g h t gains o f

>80%

were typically obtained for the

channel

proteins. Seven separate syntheses were p e r f o r m e d for Τ Μ 2 δ , whereas five 4

syntheses were c o m p l e t e d for T C a I V S 3 . 4

D i f f e r e n t cleavage protocols were used ( F i g u r e 2 H a n d I): for Τ Μ 2 δ ,


4

cleavage a n d deprotection were p e r f o r m e d i n anhydrous H F for 30 m i n at —10 °C a n d for 90 m i n at 0 °C i n the presence o f p-eresol a n d p-thioeresol, 1.4 m L / g of resin. A f t e r H F was removed, the resulting p e p t i d e - r e s i n mixture was washed w i t h anhydrous diethylether a n d d r i e d overnight u n d e r v a c u u m over K O H pellets. T C a I V S 3 was cleaved f r o m the 4

support using a l o w - h i g h H F protocol (66)

solid-phase

that considered the presence o f

tryptophane residues i n the peptide segments. T h e l o w H F step was carried out i n the presence o f dimethylsulfide a n d p-cresol for 2 h at 0 °C. H F was r e m o v e d a n d the resin was washed extensively, first w i t h diethylether a n d then w i t h c h l o r o f o r m . A standard H F protocol was then e m p l o y e d for 60 m i n at 0 °C. C l e a v e d p r o t e i n - r e s i n mixtures are stored u n d e r v a c u u m i n the dark at r o o m temperature. P e p t i d e composition a n d sequences w e r e c o n f i r m e d b y amino a c i d analysis o n a phenylthiocarbamyl ( P T C ) derivatizer-analyzer ( A B I m o d e l 420) as w e l l as automated E d m a n degradation o n a peptide sequencer ( A B I m o d e l 477A). C r u d e proteins w e r e p u r i f i e d b y reversed-phase

high-perfor

mance l i q u i d chromatography ( H P L C ) as shown i n F i g u r e 3. B o t h T C a I V S 3 4

and Τ Μ 2 δ elute f r o m the c o l u m n as well-resolved peaks. H o m o g e n e i t y o f 4

the preparation was c o n f i r m e d b y capillary zone electrophoresis (67) sodium dodecylphosphate ( S D S ) gel electrophoresis (68)

and

o n 1 6 % tricine gels

(Novex, Encinitas, C A ) . M o l e c u l a r weights ( M ) were d e t e r m i n e d using r

low-range molecular weight markers ( D i v e r s i f i e d Biotech, N e w t o n C e n t r e , M A ) . T h e molecular weight o f Τ Μ 2 δ « 11,000 a n d T C a I V S 3 « 9000. 4

4

Yields are l o w — < 1 % o f the cleaved p r o t e i n - r e s i n mixture.

Reconstitution in Planar Lipid Bilayers and SingleChannel Assay Proteins were incorporated into planar l i p i d bilayers f r o m m i x e d l i p i d - p r o tein monolayers. P u r i f i e d p r o t e i n was extracted w i t h l i p i d (Avanti B i o c h e m i cals, Alabaster, A L ) : P C [l,2-diphytanoyl-5n-glycero-3-phosphochohnel or



344


Ε tut i o n t i e e

(ain)

Ε tut i o n t i i e ( l i n )

Figure 3. Reversed-phase HPLC of synthetic channel proteins. (A) T CaTVS3 was purified by repeated reversed-phase (RP) HPLC (4 x) with a base-stable 4.6-mm X 150-mm ΡΕΒΡ-Ξ-βμ, 300-Â C-18 column (Polymer Laboratories Ltd.) equilibrated in deionized-distilled water (pH 6.5) that contained 15% Β (Β = 90% 2-propanol in water, pH 9.0). Ammonium hydroxide was used to adjust pH. The crude cleaved peptide—resin mixture was dissolved in trifluoroethanol (TFE, 99 + %; Aldrich) and filtered through glass wool. The TFE solution was adjusted to pH 7.0 with dilute NH OH. Dissolved protein was injected onto the column. Protein was eluted using the previously described gradient. Fractions were analyzed by SDS polyacrylamide gel electrophoresis (PAGE) (16% tricine; Novex). Fractions that contained the correct molecular weight species were pooled and rechromatographed as before except that the TFE solution was diluted with 10 M guanidinium-HCl followed by 2 min of sonication and 5 min of centrifugation in a microfuge. This procedure was followed for the subsequent two HPLC runs. Further HPLC (beyond 4 x) did not yield improved separation. The small peak that elutes at 52 min is also present in blank runs. (Β) T M2 8 was purified by multiple HPLC runs on a Vydac C (semiprep) 214 TP 1010 RP column equilibrated in 75% solvent A (deionized-distilled water containing 0.1% HPLC grade trifluoroacetic acid; Pierce) and 25% solvent Β (80% ν /ν HPLC grade acetonitrile and water containing 0.1% trifluoroacetic acid). Protein was purified through a series of gradient steps followed by 30-min isocratic periods at 55, 62, and 75% of solvent B. Fractions were collected and analyzed by SDS PAGE. Purified protein was reinjected onto a narrow-bore Vydac C4 RP column TP 214 54 equilibrated as previously described. Protein was eluted using the gradient 4

4

4

4

shown. POPE-POPC

[l-palmitoyl-2-oleoyl-5n-glycero-3-phosphoethanolamine

( P O P E ) a n d l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ( P O P C ) ] i n a 4:1 ratio, 5 m g / m L i n hexane (49). Bilayers were f o r m e d at the t i p o f patch pipets b y apposition o f monolayers initially f o r m e d at the a i r - w a t e r interface (69). Bilayer experiments w e r e p e r f o r m e d at 24 ± 2 ° C . E l e c t r i c a l recordings


15.

TOMICH ET AL.


345

a n d data processing were c a r r i e d out as described i n the literature (49,

69).

C o n d u c t a n c e values w e r e calculated f r o m Gaussian fits to current histograms. C h a n n e l o p e n a n d closed lifetimes were d e t e r m i n e d b y probability density analysis (69). F i g u r e 4 shows single-channel current records obtained w i t h T C a I V S 3 . 4

T h e single-channel conductance i n symmetric 5 0 - m M C a C l

2

is 7 p S . T h e

channel is cation-selective a n d conducts b o t h divalent a n d monovalent cations w i t h an apparent selectivity ratio i n f e r r e d f r o m conductance ratios o f B a Ca

2 +

> Sr

2 +

2 +

>

> N a > K r § > C I " (70, 71). C h a n n e l conductance a n d selectiv +

+

ity are i n agreement w i t h k n o w n values for the authentic D HP-sensitive


calcium channel (59). T h e D H P - s e n s i t i v e c a l c i u m c h a n n e l exhibits nanomolar affinity for m a n y D H P derivatives ( 16). I n addition, enantiomers that act as activators (agonists) or blockers (antagonists) o f c a l c i u m channels have b e e n described (17).

The

synthetic pore p r o t e i n T C a I V S 3 emulates pharmacological properties of the 4

authentic channel. F i g u r e 4 shows the effect o f addition o f the agonist B a y K 8644 ( F i g u r e 4 B ) . A d d i t i o n o f the d r u g results i n an increased channel m e a n o p e n t i m e as w e l l as an increased open-channel probability. Channels are b l o c k e d b y the D H P derivative nifedipine, as w e l l as the phenylalkylamine verapamil, the local anesthetic derivative Q X - 2 2 2 , a n d b y C d T h e remarkable stereospecific

action o f D H P enantiomers

2 +

and C a

on

2 +

.

authentic

c a l c i u m channels by the agonist a n d antagonist effects o f (—)BayK 8644 a n d (+)BayK

8644, respectively, is closely matched by the action exerted o n

T CaIVS3 4

(71).

Τ Μ 2 δ forms ionic channels i n l i p i d bilayers ( F i g u r e 5). C o n d u c t a n c e 4

events are homogeneous, and openings that last several seconds are frequent. T h e channel is cation-selective. T h e single-channel conductance i n P C bilay ers a n d symmetric 0 . 5 - M K C l is 24 p S . T h e c h a n n e l is b l o c k e d b y m i c r o m o l a r concentration o f the local anesthetic channel blocker, Q X - 2 2 2 (49). channel properties match properties characteristic of the authentic

These Torpedo

A C h R (Table I).

Sequence Specificity T h e involvement o f specific residues i n the determination o f conductance properties o f the p r o t e i n is readily addressed b y use o f the synthetic proteins. Accordingly, a tethered tetramer o f Μ 2 δ

segments i n w h i c h serine-8 is

replaced w i t h alanine was synthesized. T h i s analog, T M 2 ô ( S —» A ) , also 4

forms channels i n P C membranes w i t h a single-channel conductance i n symmetric 0 . 5 - M K C l of 20 pS (49), lower than the 24 pS observed w i t h Τ Μ 2 δ . These results are i n accord w i t h observations obtained w i t h site4

directed mutagenesis o f rodent acetylcholine receptors

(72).


346


(A)


OPEN

(Β)

Figure 4. Singh-channel recordings from a lipid bilayer containing T CaIVS3 in symmetric 50-mM CaCl . Currents were recorded at 100 mVfrom POPE-POPC membrane before (A) and after (B) addition of 100-nM racemic Bay Κ 8644. Addition of BayK 8644 results in an increase in the open-channel probability from 5% to 35% and the concurrent prolongation of the channel mean open time. 4

2



15.

TOMICH ET AL.

347


160 ms

Figure 5. Single-channel currents from lipid bilayers containing the synthetic Τ Μ2δ and T M2ô(S -» A) proteins. Currents were recorded at 100 mV in symmetric 0.5-M KCl from PC membranes containing Τ Μ2δ (À) and Τ Μ2δ (S -> A) (Β). Calculated single-channel conductances are 26 and 20 pS, respectively. For other details, see reference 49. 4

4

4

4

Table I. Ionic Conduction Characteristics of Authentic Muscle and Neuronal AChR and of the Synthetic Protein Τ Μ2δ 4

Property +

+

AChR (Neuronal)

Μ2δ Peptide

Τ Μ2δ Tetramer

50 1.1

Biomembrane Electrochemistry - ACS Publications - American

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