Use of photocrosslinkers in cell biology

MISCELLANEA

The author is at the Dept of

Biochemistry, Swiss Federal

Institute of Technology

Ztirich (ETHZ), Zurich,

UniversiMstrasse 16, CH-8092

Ziirich, Switzerland.

154

Use of photocrosslinkers in cell biology

Josef Brunner

The technique of photocrosslinkinq provides cell biologists with ‘snap shot;’ of even transient interactions between biological molecules. By UV light activation, a small photolabile group that is contained within one of the interacting molecules is converted into a highly reactive species (nitrene, carbene, radical). This attacks adjacent residues to form a stable chemical con- nection between the interacting part- ners. The products formed reflect the structural and dynamic organization of the system, as well as the chemical characteristics of the reacting molecules (for general reviews, see Refs 14).

Owing to their high reactivity, the photogenerated species can attack a wide range of chemical bonds and functions. Therefore, photocrosslinking, in general, results in chemically heterogeneous products. To trace these products and to facilitate their analy- sis, many photocrosslinking reagents and probes contain additional ‘el- ements’, such as a radioisotope (or an- other reporter group) and/or a cleavable function (see also below).

The photoactivatable groups most frequently used in the past were aryl- and nitroaryl azides. However, more recent photochemical studies raised questions regarding the general use- fulness of arylazide-based reagents (discussed in Refs 3 and 4). Among the other photoactivatable functions, the 3-trifluoromethyl-3-phenyl diazirine (TPD) group (Fig. la) combines particularly favourable properties and is now in widespread use. TheTPD group is stable under a wide range of chemical conditions (it can be manipulated without special precautions other than avoiding direct exposure to sunlight or UV) and is photolysed rapidly to a highly reactive carbene, capable of attacking even paraffinic residues. Importantly, photolysis occurs under mild conditions (long-wave UV light) that do not damage biological structures. Photolysis times depend on the light source used and, typically, range from seconds to minutes. For kinetic studies (time-dependent labelling), the TPD group can also be activated with a short (cl ms) flash from a powerful xenon flash-tube. In general, the most relevant factor in photocrosslinking is the lifetime of the

0 1996 Elsevier Science Ltd PII:SO962-8924(96)40001-O

reactive intermediate, which, for the carbene generated from TPD, is presumably in the nanosecond range (at ambient temperature). This article fo- cuses on recent developments in which the unique properties of the TPD group are exploited to provide information on the structural and functional organization of biological systems.

Probes of membrane structure and function

The first TPD-based reagent that found widespread application was 3- trifluoromethyl-3-(m-[1251]iodophenyl) diazirine ([1251]TID; Fig. 1 b; Ref. 5). Owing to its highly apolar character, [12sl]TID partitions into the lipid core of membranes and, therefore, labels selectively those portions of integral proteins that are embedded in the lipid bilayer (reviewed in Ref. 2). Over the past ten years, a limited number of soluble proteins have also been identified that can be labelled with [1251]TID. Probably, these proteins possess hydrophobic regions with a certain affinity for this reagent. In the case of the nicotinic acetylcholine receptor from Torpedo California, [’ 251]TI D acts as a potent noncompetitive an- tagonist, suggesting that it binds specifically to a distinct, presumably hydrophobic, pocket within the re- ceptor6. An unpleasant aspect of the use of [1251]TID is its volatility. Since hydrophobic labelling of integral proteins is rather inefficient (typically, only l-2% of the total reagent associates with proteins), comparatively large amounts of the volatile radioactive reagent are needed. Fortunately, sev- eral phospholipid-based reagents are now available (see below), which, be- sides being nonvolatile, offer other advantages.

Based on a new, modular design, a wide array of radioiodinated reagents can now be conveniently prepared with very high specific radioactivity7. In this design, the reagents are prepared as nonradioactive, tin-containing precursors that in a simple, final step can be converted into the desired radioiodinated molecules. Reagents of high specific radioactivity are indispensable where the target component is present only in minute quantities, as is the case, for example, for many receptors.

Using this new approach for synthesis, a number of reagents (Fig. 1 b) have been prepared, all at a specific radioactivity >2000 Ci/mmol. [1251]TID-benzoic acid ester ([1251]TlD- BE) is an improved, less volatile version of [1251]TID. The other reagents are all structural analogues of lipids, which, as detailed below, are finding application as probes of membrane topog- raphy and as photoaffinity-labelling reagents.

In addition to being completely nonvolatile, [1251]TID-phospholipids offer other advantages over reagents such as [1251]TID or [1251]TID-BE. The long-chain lipid [1251]TID-PC/1 6 combines particularly favourable properties. Once incorporated into a lipid bilayer (by reconstitution or by lipid exchange mediated by phospholipid exchange proteins8), it does not un- dergo rapid spontaneous exchange or transfer between membranes. Thus, [1251]TID-PC/1 6 is an ideal reagent to use in the investigation of vectorial systems and processes involving more than one membrane. To illustrate this point, it has long been known that H+-induced activation of the fusion capacity of haemagglutinin (HA) from influenza virus causes exposure of the fusion peptide, a hydrophobic segment with membrane-binding activ- ity. However, it was unclear whether the anticipated interaction of the peptide with lipids involved the target and/or the viral membrane. By labelling HA with [1251]TID-PC/1 6, present either in the viral or target membrane bilayer, it has now been found that, during the initial binding step, the fusion peptide associates with the target membrane, whereas later in fusion, or during H+-induced virus in- activation, the peptide penetrates the viral membrane8. That the fusion peptide can insert into the target membrane is consistent with conclusions from recent studies of the structure of HA at pH 5 (Refs 9 and 10). The abil- ity of the fusion peptide to insert also into theviral membrane, although not unexpected”, is more difficult to ex- plain and may reflect an inherent property of HA that is relevant for fu- sion8,12. Labelling with [1251]TID-PC/1 6 has also led to the identification of a putative fusion segment in the ecto- domain of the major glycoprotein of rhabdoviruses13. An interesting finding was that this membrane-active segment, unlike most other known fusion peptides, is not rich in hydrophobic amino acids. A similar situation may exist for the major myristoylated alanine-rich protein kinase C substrate

trends in CELL BIOLOGY (Vol. 6) April 1996

(MARCKS). Again, as concluded from [12sl]TID-PC/1 6-labelling, association of MARCKS with membranes involves an internal, possiblyamphipathic, polypeptide segment, in addition to its N-terminally-linked myristoyl residue14. Considering the growing list of proteins whose activities are controlled by the reversible association with membranes, labelling with [12sl]TID-PC/1 6 and related lipids will find further useful applications.

Lipids are not merely the main components of membranes, but also play a variety of distinct functional roles. Importantly, they are precursors of second messengers that are generated in cells following stimulation of surface receptors. The identification of the various cellular targets of these lipid second messengers is an important objective of current research. There is growing evidence suggesting that carbene-generating analogues of lipid second messengers make powerful photoaffinity probes. This is exempli- fied by the recent specific labelling of the putative membrane receptor for lysophosphatidic acid (LPA) by using a 32P-labelled diazirine-LPA (Ref. 15). Ongoing experiments employing [12sl]TID-ceramide to identify the cellular targets for ceramide, a lipid second messenger generated upon cytokine stimulation of cells, are also proving informative (j. Pfeilschifter and S. Schtitze, pers. commun.). Labelling with [1251]TID-ceramide is also being used to probe the role of sphingolipids in membrane fusion induced by Semliki Forest virus. This compound may also prove useful in elucidating the anticipated function of sphingolipids in the sorting of GPI-anchored membrane proteins.

Peptide- and protein-based photocrosslinkers

To introduce a photoactivatable group into a polypeptide chain, three principal techniques can be used: chemical modification of reactive amino acid side-chain residues, and both chemical and translational incorporation of a photoactivatable amino acid during peptide-chain elongation.

Protein-based probes are prepared generally by specific chemical modification of a nucleophilic amino acid side-chain function (e.g. an SH- or NH,-group) with a heterobifunctional reagent containing a conventional reactive group on one end and a photoactivatable group on the other. Many of these reagents are cleavable, and some, in addition, contain a radioisotope. As illustrated in Figure 2(a),

MISCELLANEA


(a)

N=N

(b)

[“51]TID

F3;p 125, J n = 4 [‘=l]TID-PC/6

W,), 0

?-

i

0-~-0L4(CH3)3 n = IO: [“51]TID-PC/12

~,cW’,)ny~ 0- n = 14: [‘*5l]TID-PC/16

0

125, F3;@ x W,), p

&O-CHp<;;-

0- 3+

H~C’(C~$O [‘251]TID-PS/1 6

0 OH

OR 125,

0 i iH

R = H: [1251]TID-ceramide

FIGURE 1

(a) Scheme of the photolytic decomposition of 3-trifluoromethyl-3-phenyl diazirine (TPD). Reactions of the photogenerated singlet carbene with various functional groups are shown. (b) Chemical structures of 3-trifluoromethyl-3-(m [1251]iodophenyl) diazirine ([1251]TlD) and various [12SI]TID-ester based photo(affinity) labelling reagents. The full chemical names of the compounds are: 2-[125l]iodo-4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl benzoate ([‘251]TID-BE); 1 -O-hexanoyl-2-0-[9-[[[2-[125l]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)- benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphochoIine ([1251]TID-PC/6); 1-0-dodecanoyl-2-0-[9-[[[2-[‘251]iodo-4-(trifluoromethyl-3~-diazirin-3-yl)- benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine ([1251]TID-PC/1 2); 1 -O-hexadecanoyl-2-0-[9-[[[2-[125l]iodo-4-(trifluoromethyl-3H-diazirin-3-yl) benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine ([1251]TID-PC/1 6); 1 -O-hexadecanoyl-2-0-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)- benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphoserine ([‘2sl]TID-PS/1 6); and N-[3-[[[2-[12sl]iodo-4-(3-trifluoromethyl-3~-diazirin-3-yl)- benzyl]oxy]carbonyl]propanoyl]-o-erythro-sphingosine ([1251]TID-ceramide).

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(a) Target

molecule

+ Photo- crosslinking

0

i - -NH-CH-CO- -NH-CH-CO-

Cleavage * +

T -NH-CH-CO-

(b)

[‘a51]TID-M3

~J~+N$ [‘25l]TID-OSu/4

F3C 0 N-N

FIGURE 2

(a) Principle of ‘label transfer’ crosslinking. The side chain of an amino acid within a polypeptide chain contains a photoactivatable group (filled circle), a radioisotope

(asterisk) and a cleavable function (pair of triangles). After covalent

photocrosslinking to a target molecule, the susceptible bond is cleaved, whereby the peptide chain is liberated and the radioisotope transferred onto the target

component. (b) Chemical structures of 3-maleimidopropionic acid 2-[‘25l]iodo-4-(3-trifluoromethyl-3Kdiazirin-3-yl)benzyl ester ([1251]TlD-M/3) and

3-[[[2-[125l]iodo-4-(3-trifluoromethyl-3H-diazirin-3-yl)ben~l]oxy]carbonyl]propanoic acid, N-hydroxysuccinimide ester ([12sl]TlD-OSu/4). Both reagents contain an ester

linkage that is cleaved easily by treatment with a nucleophile such as hydroxylamine or ammonia.

this serves to facilitate the identification of photocrosslinked target molecules. Two new TID-based ‘label transfer’ photocrosslinking reagents (Fig. 2b) have been added recently to the growing list of bifunctional crosslinking reagents. Whereas the N-hydroxysuccinimide ester [1251]TID- OSu/4 can be coupled to primary or secondary amines, the electrophilic end of [1251]TID-maleimide ([1251]TlD- M/3) reacts preferentially with sulf- hydryl groups. An obvious compli- cation of this approach is that, in general, modification of protein can occur at multiple sites.

The steric and functional pertur- bation that may result from modification of a side-chain residue can be minimized by directly incorporating into the polypeptide chain a photoactivatable amino acid, such as L[3-(trifluoromethyl)-3-diazirin-3Kyl] phenylalanine [(Tmd)Phe; see Fig. 3 for structure of (Tmd)Phe residue; Refs 16 and 171. (Tmd)Phe is fully compatible with the Fmoc or Boc strategy of chemical peptide synthesis. Peptides with such ‘built-in’ photoactivatable

156

residues are finding increased application in the identification of receptors for peptides and for mapping the cor- responding binding sites (for an example, see Ref. 18).

The first ribosome-catalysed incorporation of an amino acid carrying a photoactivatable group was reported in 1986 (Refs 19 and 20). In these studies; an in vitro translation system (wheat germ) was supplemented with a modified lysyl-tRN’A whose lysyl e-amino group had previously been acylated with azidonitrobenzoic acid. This approach proved excep- tionally powerful in studying co- translational insertion of proteins into the membrane of the endoplasmic reticulum21.

A more versatile method uses a tRNA chemically charged with the nonnatural amino acid (Tmd)Phe. In a first step, the N-protected amino acid is acylated to the dinucleotide p(d)CpA (rather than to tRNA). The resulting 3’(2’) (amino)acyl-p(d)CpA is then ligated to an ‘abbreviated’ tRNA lack- ing the terminal bases pCpA at the 3’-acceptor end 22-28. To incorporate

the nonnatural amino acid at a single, selected position with& the growing polypeptide chain, it is linked to suppressor tRNA which will insert its amino acid into the polypeptide in response to a stop codon in the mRNA (Fig. 3). Functional suppressor tRNA need not be isolated from natural sources, but can be generated easily by the run-off transcription of a corre- sponding DNAtemplate24,25.Although such artificial suppressor tRNAs lack modified bases, suppression efficiencies are comparable to those obtained with suppressor tRNA prepared from yeast tRNAPhe by the anticodon-loop- replacement technique2g.

As discussed in a review in this issue30, crosslinking, employing polypeptide probes generated in vitro containing the (Tmd)Phe group, provides significant new insight into the mechanism of protein insertion and translocation into and across the membrane of the endoplasmic reticu- lum31,32. From a technical perspec- tive, the efficient crosslinking of a (Tmd)Phe residue within the signal sequence of the invariant chain to fatty acyl residues of membrane phospholipids is particularly gratify- ing as it illustrates, once more, the exquisite reactivity and generally low chemical selectivity of the photogenerated carbene. The marked differences in efficiency in which nascent chains are crosslinked to various protein components are thought to reflect differences in the tightness of the underlying interactions (weak interactions are likely to favour unpro- ductive quenching reactions of the photogenerated carbene with water or buffer components), in the orien- tation of the (Tmd)Phe side chain, as well as in the intrinsic reactivities of the target-site residues (even car- benes display a certain degree of chemical selectivity). Yet, in spite of the fact that many details of the chemistry of photocrosslinking are only defined poorly and only few studies have been completed, there remains little doubt that polypep- tides generated in vitro containing a (Tmd)Phe will prove similarly useful in many other areas; for example, for probing ‘later’ steps, such as protein folding and oligomeric assembly (interactions with chaperones), and sorting events. Also, site-specific photocrosslinking has been used successfully to study insertion of newly synthe- sized proteins into the endoplasmic reticulum of permeabilized cells33, and this technique may even be ap- plicable to intact cells28.


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Amber suppressor

tRNA

5’ ______( UAG j 3’ mRNA

Codon

In vitro translation

Mutant polypeptide with (Tmd)Phe site-specifically incorporated

FIGURE 3

Scheme representing the decoding step in the protein translation that leads to

site-directed incorporation of L-[3-(trifluoromethyl)-3-diazirin-3H-yl]phenylalanine

((Tmd)Phe) into polypeptide chains. Translation in vitro is performed in the

presence of an amber suppressor tRNA chemically aminoacylated with (Tmd)Phe. The amino acid is incorporated in response to an amber (UAC) stop codon engineered within the coding region of the mRNA.

Concluding remarks Although still largely unexplored,

the range of potential application of the recently developed reagents and experimental approaches for photocrossiinking is immense. Photo- crosslinking is a powerful, flexible and informative method that is simple and, once the reagents are available, does not require expensive equip- ment. The design of an experiment, the choice of the reagent(s) and, no- tably, the strategy to detect and analyse crosslinked components are determined largely by the specific questions to be answered.

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trends in CELL BIOLOGY (Vol. 6) April 1996 157

Use of photocrosslinkers in cell biology

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