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Synthesis of Biological Macromolecules Chemical Methods for the Generation of Large Polypeptides HIV-1 Protease as a Paradigm for Elucidating Biological Function by Chemical Protein Synthesis Semisynthetic Proteins of the Ras-Superfamily Split-Inteins for Protein Semisynthesis in vitro and in vivo Prospects
Max-Planck Institut
The enormous progress made in the use of gene technological techniques over the past several decades has been the main driving force in the accumulation of our knowledge of biology at the molecular level. This progress has at times tended to push more classic approaches, such as those stemming from synthetic chemistry, into the background, and there has even been a tendency to regard contributions from this area as being superuous. This attitude has begun to change recently, with the emergence of the eld now referred to as chemical biology, and it is now appreciated that synthetic chemistry can make a unique contribution to the outstanding problems in fundamental biological and medically oriented research. The full potential of these methods is beginning to be realized in the area of peptide and protein synthesis, and this will be the topic of this article.
ca. 50 nucleotides is relatively facile on a solid support, and that enzymes can be used to ligate such fragments in a directed fashion to achieve the goal of total gene synthesis. Although this is not the most routinely used method for generation of complete coding regions for specic proteins, there are often situations where this is the method of choice, because it allows complete control of codon usage to optimize protein expression in the organism to be used. Gene synthesis is now offered on a commercial basis and plays a signicant role in modern biological research. Progress toward total synthesis of proteins has been slower, mainly due to the lack of easy availability of an enzymatic procedure equivalent to DNA ligation that would allow coupling of peptides of a length that can be conveniently prepared by solid-phase synthesis (depending on sequence, the largest fragments that can be produced are between 50 and 100 residues long). This situation has changed signicantly over the past 1015 years with the introduction and widespread use of methods for the ligation of protein fragments together with the combination of the methods of synthetic chemistry with techniques originating in biology. In the following, we initially discuss the advances that have been made at the technical level, and then introduce some of the many applications that exploit the new methods for the study of biologically important processes. 1
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of chemical synthesis to the production of functional as well as site-specically modied enzymes concerns the protease from human immunodeciency virus 1 (HIV-1 PR). This enzyme cleaves the gag-pol polypeptide into functional proteins during virion budding from host cells and is essential for replication of the virus (12). Inhibitors of HIV-1 PR are an important class of anti-HIV drugs, and their development is at least partially based on the availability of structural and molecular information obtained with chemically synthesized HIV-1 PR.
HIV-1 Protease as a Paradigm for Elucidating Biological Function by Chemical Protein Synthesis
Chemical protein synthesis and semisynthesis have been used to study the molecular basis of protein function in numerous cases. One of the very early and most impressive applications 2
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O SH
OH HO O SR + H2N HO HS O NH Peptide 2
O H Peptide 2 HO
H2N
NH
O SH
OH SH O OH Peptide 2 O HO O NH
Peptide 2 HO
N H
NH2
H 2N
NH
Figure 1 Native chemical ligation (NCL) between two unprotected peptide segments. The initial transthioesterication reaction leads to an intermediate that undergoes an S to N-acyl shift via a ve-membered cyclic transition state and generates a native amide bond at the ligation site.
is due to the positioning of the two glycine residues on the outside of the aps, away from the substrate. However, the synthesis of another HIV-1 PR analog by Kent and Baca placed the thioester bond between Gly49 and Ile50, leading to a reduction in catalytic activity by a factor of 3000 (Fig. 2b) (22). This constituted the rst experimental evidence that hydrogen bonds between the backbone of the ap region and the substrate are important for catalytic activity. However, substrate specicity and afnity were not affected. These particular hydrogen bonds are transmitted from the protease backbone to the substrate via an internal water molecule and are believed to contribute to the distortion of the scissile bond of the substrate (23). The applicability of the thioester-forming chemoselective ligation approach was broadened by the fact that this chemistry can be carried out under acidic conditions in the presence of sulfhydryl groups. By taking advantage of this selectivity of the
alkylation reaction, two different HIV-1 PR monomers were prepared. These monomers carried a free sulfhydryl group at their N- or C-terminus, respectively, and were, subsequent to the thioester-forming ligation step, joined together by a disulde linkage to generate tethered dimers of two distinct HIV-1 subunits (24). This tethering of the two subunits produced one of the largest functional proteins prepared by chemical synthesis at that time and allowed the preparation of HIV-1 PR molecules with asymmetrically placed subunits. One example of such asymmetrical HIV-1 PR analogs was constructed with one subunit having a thioester bond between Gly51 and Gly52, which did not interfere with the biological activity of the protease, and a subunit that had a thioester bond between Gly51 and Gly52 and an additional ester bond instead of an amide bond between Gly49 and Ile50 (23). By replacing an amide with an oxygen atom in a unique position, no backbone hydrogen 3
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(a)
O
HIV-1PR 1-51
(b)
O
HIV-1PR 53-99 HIV-1PR 1-49 HIV-1PR 51-99
SH + Br O
SH + Br O
Thioester-forming Ligation O
HIV-1PR 53-99 HIV-1PR 1-51
O
HIV-1PR 51-99 HIV-1PR 1-49
S O
Figure 2 Chemical synthesis of backbone engineered HIV-1 protease. The peptide segments were synthesized by SPPS and harbored a C-terminal thioacid (HIV-1 PR, aa 1-49/51, shown in blue) or an N-terminal bromoacetic acid modication (HIV-1 PR51/53-99, shown in red). These unique functional groups lead to an unnatural thioester bond either between Gly51 and 52 (Strategy A; the ligation site is shown in yellow in the cartoon representation of the HIV-1 PR dimer) or between Gly49 and Ile50 (Strategy B; the ligation site is located at the end of the N-terminal peptide segment depicted in blue). The chemoselective ligation reaction is followed by purication steps and folding of the protein into its functional conformation. Strategy A led to a fully functional HIV-1 PR, whereas strategy B led to a severe reduction in catalytic afnity. The functional dimer of the HIV-1 PR is drawn as a cartoon with only one subunit showing the modications introduced during synthesis. The second subunit (in green) is shown unmodied for clarity. Aspartic acid 25, site-specically labeled with 13 C for NMR spectroscopy studies, is shown in magenta in one HIV-1 PR subunit.
bond to a substrate carbonyl (via a water molecule) can be formed. Therefore, such a construct should exhibit a highly reduced catalytic activity if both ap regions are required to form hydrogen bonds. However, the ester analog of HIV-1 PR showed a reduction of kcat by only a factor of 2 upon this atom replacement. This demonstrated that only one ap region is used by the enzyme for catalysis and that the slightly reduced enzymatic activity of the ester analog is caused by the fact that, in such an asymmetric dimer, only one substrate orientation leads to productive binding. This is a vivid example of chemical protein synthesis as a unique tool in the quest of elucidating the molecular basis of enzyme catalysis.
13 C
as a function of the pH and the presence and absence of substrate or inhibitor molecules. These titration experiments provided additional evidence for the suggested working model of aspartyl proteases and conrmed that HIV-1 PR is a member of this class of enzymes (26). The two aspartyl side-chain carboxyl groups (one from each subunit) act as general base and acid, respectively, thereby leading to the breakdown of the enzyme-substrate intermediate. The work on HIV protease demonstrates how chemical protein synthesis allowed isotope labeling of a 22-kDa protein with atomic precision and provided further insights into the chemical basis of the proteolytic cleavage reaction. Isotope labeling with atomic precision has since then been used to reveal structural features of other either chemically synthesized or semisynthetic proteins (2729).
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problem of generating a C-terminus that is either naturally or unnaturally modied. In one of these, C-terminal peptides have been linked by a chemical method leading to an unnatural link. The chemistry used was based on the reaction of a truncated protein carrying a C-terminal cysteine with peptides carrying an N-terminal -maleimidocaproyl group (3337). In this manner, Ras derivatives containing C-terminal lipids (farnesyl in the case of K-Ras and farnesyl and palmitoyl in the case of H- and N-Ras) could be prepared as well as those containing uorescent or reactive groups. The most important result to emerge from these studies concerns the reversible modication of a cysteine residue by palmitate (38). Ras proteins seem to display weak and nonspecic general interactions with membranes via their farnesyl group (or a polybasic domain in K-Ras), but they are palmitoylated on Golgi membranes leading to their capture here. From there, they can be shuttled or reshuttled to their location on the plasma membrane by vesicular transport. This specic localization at the Golgi and plasma membranes did not occur when the palmitoyl group was replaced by a stable hexadecyl thioether, thus demonstrating the importance of a cycle of acylation and deacylation in the mode of action of these proteins. In the example described, which uses chemistry to create an unnatural linkage between the C-terminal region of Ras and the rest of the protein, there was no apparent detrimental effect of this departure from the natural peptide backbone, as shown by various tests of biological activity. This is presumably because the most important function of the region in the experiments discussed is to provide a exible linkage to the lipidated terminal residues. In other cases, there is a reason to believe that such a modication might be less well tolerated. In the case of the Rab proteins, which are members of the Ras-family involved in the regulation of vesicular transport, it is clear that the exact structure (sequence) of the hypervariable C-terminus is of critical importance for directing the individual members of the family of over 60 Rab proteins to distinct membrane targets. For this reason, and because one question to be investigated involved structural studies on complexes between Rab proteins and their partners, a method for producing posttranslationally modied Rab proteins with a natural polypeptide backbone throughout the whole protein was needed. This was achieved using the technique of expressed protein ligation (EPL), a procedure introduced by Muir et al. (3941). The procedure has been used in the Rab eld for the construction of a number of C-terminally modied proteins which have been used in biochemical, biophysical and cell biological studies (4246). In a specic case, as shown in Fig. 3a, a yeast Rab protein, Ypt1, was expressed in C-terminally truncated form in E. coli as a fusion protein with an intein domain and a chitin-binding domain (46). This construct could be puried by afnity chromatography on chitin-agarose. The C-terminal thioester of the truncated Ypt1 was cleaved from this support using a thiol reagent, a procedure that emulates the attack of a serine or cysteine residue in the C-extein, which is normally present in natural intein proteins (47). This thioester could be used for an in vitro ligation reaction with monogeranylgeranylated di-cysteine to generate the C-terminus in monolipidated form. As both the prenylated peptide and the reaction product (prenylated Ypt1) are insoluble 5
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S O
SO3
HS + H2N O
Transthioesterification
S
O H2N
N-acylshift
H N O
SH
(b)
Figure 3 (a) Preparation of prenylated Ypt1 (a yeast Rab-protein) by expressed protein ligation. A C-terminal thioester of the truncated Rab protein was allowed to react with a doubly geranygeranylated tricysteine peptide, leading to transesterication and an SN acyl shift to generate a native peptide bond. (b) Interaction of the C-terminus of semisynthetic doubly geranylgeranylated Ypt1 with the lower domain of yeast GDI. GDI is shown in green as a ribbon structure, the C-terminus of YPT1 in magenta, and the geranylgeranyl groups in red and blue CPK representation. Several residues of the C-terminus of YPT1 were not visible in the electron density map, so that the connection to the prenyl groups is not observed directly. One prenyl goup (in red) is buried deeply into the hydrophobic core of GDI, whereas the other (in blue) is more supercially bound and shows interaction with the other prenyl group. The lipid binding site is generated by an opening movement of two -helices.
in an aqueous environment, the ligation reaction was performed in detergent solution. Using the expressed protein ligation approach, both singly and doubly prenylated Ypt1 molecules could be produced. The complexes of these proteins with their solubilizing protein, GDI (GDP-dissociation inhibitor), could be 6
crystallized, and their three-dimensional structures were determined (4648). This revealed for the rst time the nature of the lipid interaction with a binding site in an unexpected part of the GDI molecule (Fig. 3b). In the previously determined structure of GDI without a bound Rab molecule, this binding site was not detected, because a movement of one of the -helices of
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the lower domain of GDI has to occur to create space for lipid binding, and this seems only to occur when the lipid residues, or possibly the whole prenylated Rab molecule binds. The position of binding was essentially the same for single or double geranygeranyl groups, and Fig. 3b shows only the physiologically more relevant doubly prenylated structure (most Rab molecules are doubly prenylated). The structural determination of the complex between GDI and prenylated Rab molecules has provided considerable information on the mechanism of action of GDI in the recycling of Rab proteins between target and donor membranes (48, 49). It also sheds light on the molecular basis of a form of x-linked non-syndromic mental retardation, in which there is an L92P mutation in GDI, which is highly expressed in brain, and which results in a reduced ability to extract Rabs from membranes. It was previously thought that this residue would be in the lipid binding site, but the structure depicted in Fig. 3b shows that the corresponding residue in yeast GDI (I100) is not in the lipid binding site but makes an important hydrophobic interaction with a conserved hydrophobic motif in the Rab C-terminal hypervariable domain. The same technology was used to create Rab proteins bearing a variety of uorescent groups at the C-terminus. This approach allowed introduction of such reporter groups near to the reactive SH groups, which are the site of prenylation while leaving these groups free for the prenylation reaction, a process that results in large uorescence signal changes in certain cases. Experiments on the prenylation of such selectively modied Rab proteins allowed insights into the molecular basis of another hereditary disease, namely x-linked degradation of chorioretinal cells in choroideremia, a disease caused by underprenylation of certain Rab proteins (50).
consists of only 36 aa and is easily accessible by chemical synthesis and therefore allows the addition of specically modied peptides to its C-terminus that are, upon trans -splicing, transferred onto the N-terminal protein that was expressed as a fusion protein with the N-terminal intein segment. This split intein system has enabled the rst semisynthesis of a GFPFLAG fusion protein in vivo (59). To achieve this goal, the N-terminal DnaE segment was fused to GFP and expressed in CHO cells. These cells were complemented with a chemically synthesized C-terminal part of the intein together with a FLAG tag and a protein transduction domain (PTD) for efcient uptake into the cells. The GFPFLAG fusion protein that was generated upon successful trans -splicing was unambiguously identied by GFPand FLAG-specic antibodies. Such a system allows the in vivo incorporation of biophysical probes, as long as the chemically synthesized part can be brought into the cells of interest. Detailed insights into the mechanism of the trans -splicing reaction of the DnaE intein were provided by crystal structures of this protein after excision and of a splicing-decient precursor protein (60). Further applications of the DnaE split intein include the development of a tandem trans -splicing system that is based on a combination of the DnaE split intein and the engineered, inducible VMA split intein (61). Such a system allows the segmental labeling of proteins with specic isotopes [as demonstrated by Otomo et al. with the articial PI-Pfu I and PI-Pfu II split inteins (62)] and uorophores. The DnaE split intein was also used by Camarero et al. to achieve the site-specic, oriented immobilization of proteins such as maltose binding protein (MBP) and enhanced green uorescent protein (EGFP) onto glass surfaces (63). A covalent bond to the glass surface was established by thioether formation between a maleimide group on the surface and a thiol group bearing PEG linker that also carried four amino acids, including a cysteine residue, which could act as a nucleophile in trans -splicing reactions, and the C-terminal segment of the DnaE intein (36 aa). Upon addition of a MBP- or EGFP-N-intein fusion construct that was either produced by recombinant or cell-free expression the intein halves associated and trans -splicing occurred, leading to the immobilization of MBP or EGFP on the surface. The associated DnaE intein halves were washed away, and the proteins remained, covalently bound via a PEG spacer, on the surface. The advantage of this approach is that no purication of the expressed proteins is necessary because only intein fusion constructs undergo the highly specic immobilization reaction. Furthermore, only low concentrations are needed to achieve efcient trans -splicing reaction [dissociation constant of the DnaE split intein halves is 43 nM, and trans -splicing occurs at a rate of ca. 7 105 s1 (61, 64)], which constitutes an advantage over immobilization techniques that rely on chemoselective reactions and strongly depend on reactand concentrations (6568). Thus, this approach points to a new route to produce protein chips without the need for large amounts of puried protein.
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O H N-Extein
HS O N H N-Intein + C-Intein H N
HS N H C-Extein
O NH2
O NH2
O NH O + H N-Extein
HS N H
C-Extein
Figure 4 Mechanism of trans-protein splicing. (a) Initial association of the intein halves to form a functional intein. (b) Activation of the N-terminal splice-junction via an N-S acyl shift. (c) Formation of a branched intermediate upon transthioesterication. (d) Branch resolution and intein release by succinimide formation. Spontaneous SN acyl rearrangement yields the processed product with a native peptide backbone.
a functional mini intein (154 aa) that can also be split into two halves that undergo trans -splicing when co-expressed in E. coli (57). To test whether trans -splicing also occurs in vitro , Mootz et al. expressed a fusion protein consisting of MBP and the N-terminal half of the DnaB intein (104 aa) and a fusion construct of the C-terminal half (47 aa) and a hexa-histidine tag (69). Upon mixing in stoichiometric amounts, successful trans -splicing produced the MBP-His-tag fusion protein. This constituted the rst case of an articial split intein that spontaneously assembled to form the active intein and underwent trans -splicing without the need for a denaturationrenaturation step. The only other articial split intein that does not require such a renaturationdenaturation step reported previously was the VMA intein from Saccharomyces cerevisiae . However, the N- and C-terminal segments of this intein do not assemble 8
spontaneously to form a functional intein. They require a dimerization domain that brings both halves in close proximity to each other, which induces trans -splicing (7072). This renders the DnaB split intein highly interesting for protein engineering approaches, and in combination with the DnaE split intein or with an inducible split intein such as the VMA intein, it provides a valuable tool to combine three protein segments with each other by two concomitant or subsequent trans -splicing reactions. An additional advantage of the DnaB split intein is the occurrence of a serine residue as the C-terminal nucleophile for the splicing reaction instead of cysteine residues. Cysteine residues might not be desirable in some cases because they can interfere with folding or labeling of the newly generated protein. Nevertheless a cysteine can replace the serine as a nucleophile at this position as demonstrated by the fact that the DnaB intein has been used to generate protein segments with N-terminal
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cysteine residues. This was achieved by expressing the desired DnaB intein as a fusion construct with the target protein in inclusion bodies and by taking advantage of the pH sensitivity of the DnaB intein to prevent premature cleavage during work up (73). To extend the utility of the DnaB split intein, Liu et al. have tested 13 different sites to split this intein into two segments of different length (58). Until this series of experiments, all known articial split inteins had been split at the endonuclease domain. Out of 13 tested sites, 3 gave functional split inteins that would undergo trans -splicing, including 1 that consisted of only 11 N-terminal amino acids. Such a short N-terminal split intein half is accessible by chemical synthesis, and the introduction of chemically modied peptides at the N-terminus via trans -splicing was recently demonstrated. Such a system nicely complements the already established C-terminal modication approach via the DnaE split intein (74).
Prospects
The work reviewed here illustrates that, in the century since Fischer formulated his vision that the synthesis of proteins should be achievable using the methods of organic chemistry (1) this prediction has been largely fullled. What he could not possibly have predicted was the role that molecular biological techniques would play in combination with chemical methods, although he was realistic enough to imply that chemistry would not be the method of choice if biotechnological methods were available. Future developments in the area of synthetic and semisynthetic proteins are likely to include extension of ligation methods to amino acids other than cysteine and the increased use of strategies for generating proteins with precisely engineered properties in cells, including such approaches as conditional splicing, a technique in which a specic protein activity is generated intracellularly by exposure to a small membrane-permeable molecule (7072).
References
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See Also
Chemistry and Chemical Reactivity of Proteins Structure, Function and Stability of Proteins Lipidated Peptide Synthesis Synthesis of Natural and Unnatural Amino Acids
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
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