3.7: Examples Visualization- LibreFest 2000 Molecular Visualization (change back to Peptides and Proteins) - Biology

3.7: Examples Visualization- LibreFest 2000 Molecular Visualization (change back to Peptides and Proteins) - Biology

We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

3.7: Examples Visualization- LibreFest 2000 Molecular Visualization (change back to Peptides and Proteins)

Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTT NT ) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTT NT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTT NT , in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTT NT cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both HTT NT and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide and have implications for the molecular mechanism of Huntington's disease.

Access options

Get full journal access for 1 year

All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.

Get time limited or full article access on ReadCube.

All prices are NET prices.


Since antibiotic resistance is still an increasing health problem, investigation of new antibiotics such as cationic antimicrobial peptides (AMPs) is of great interest. Different than non-peptide-based antibiotics, which usually inhibit cell wall and protein biosynthesis or DNA replication, AMPs act predominantly without binding to specific receptors but interact directly with the lipid matrix of bacterial cell membranes [1]. Cationic peptides target mainly negatively charged lipids exposed on the surface of bacterial membranes. We recently reported the different effects of lactoferricin-derived peptides, nonacylated and N-acylated, on E. coli membranes and on model liposomes composed of bacterial lipids [2]. Perturbation of the lipid organization in liposomes composed of negatively charged lipids such as phosphatidylglycerol or total E. coli lipid and binding to lipopolysaccharides (LPS) were shown to be enhanced by hydrophobic modifications of the peptide by N-acylation. However, zwitterionic lipids such as phosphatidylcholine or phosphatidylethanolamine (PE) were also partially affected by such peptide derivatives [2] [3]. The cell envelope of Gram-negative bacteria, like E. coli, consists of a cytoplasmic or inner membrane (IM), a peptidoglycan layer, and an outer membrane (OM) [4]. The OM itself harbors proteins, phospholipids and LPS [4]. A number of AMPs (e.g. tachyplesin, magainin and cecropin A) first interact with the negatively charged LPS to form a complex [5], then transfer across the OM to the periplasmic space [6], and finally perform their lethal effect by interaction with the IM, which is mainly composed of PE, PG and cardiolipin (CL) [7]. Numerous studies demonstrated that AMPs interfere with the integrity of bacterial membranes via diverse mechanisms (for reviews see [5] [8] [9]). The most frequently discussed modes of action include the formation of toroidal pores [10] [11] and the coverage of the membrane surface by peptides (carpet model [12]). The capability of AMPs to cluster anionic lipids is also described to be a mechanism applied by cationic peptides [13]–[15]. However the mechanism of the final killing step appears to depend on peptide concentration and type [16] as well as on their structure in presence of membrane [17]–[20]. For instance, it was shown that the structure of the antimicrobial center of bovine lactoferricin (LFcinB - RRWQWR-NH2) bound to sodium dodecyl sulfate (SDS) micelles is amphipathic with the Trp side chains separated from the Arg residues [19]. At concentrations below the minimum inhibitory concentration (MIC) membrane blebbing and detachment of IM and OM were observed in the presence of peptide LL-37 and cecropin B, while membrane lysis of E. coli occurred at their MIC [16]. In the case of human lactoferricin (hLFcin) derivatives, we previously reported detachment of OM and IM and protrusions at MIC, and established that this severe membrane effect led to bacterial killing before visible membrane lysis [2]. Fragments of LFcinB were shown to localize in the cytosol of E. coli [21], where they affected protein and DNA synthesis [22]. After one hour of exposure to LFcinB at MIC, a profound effect on cell morphology of E. coli was observed. The LFcinB exposed cells became filamentous and elongated and did not appear to be dividing in a regular manner [22]. Similar effects were observed when E. coli was incubated with the antibiotic bicyclomycin, which inhibits septum formation and converts the cells to filamentous forms. The antibiotic also induced high undulation and numerous blebs of the outer membrane [23]. Such formation of filamentous E. coli was also observed in the present study though only in the presence of N-acylated peptides.

Besides proteins that have been reported to be required in the process of cell division and septum formation in prokaryotes [24] [25], the specific involvement of phospholipids in this membrane-associated process has also been investigated [26]–[29]. For example, PE was proposed to play an important direct or indirect role at some stage of the cell division cycle, since the E. coli mutant pss-93, which lacks PE grows as filamentous cells and is apparently defective in cell division [27]. In this mutant FtsZ rings localized properly at division sites but failed to constrict [27]. PE is a lipid able to undergo a bilayer-to-non-bilayer transition, a property that might be essential in cell division processes [30] [31]. Similarly CL is reported to play a role in cell division, e.g. via formation of membrane domains that seem to participate in this process [28]. However, though CL is also able to undergo a bilayer-to-non-bilayer transition in the presence of divalent cations, it was shown not to completely compensate for PE in the PE lacking mutant pss-93 [32]. To gain further insight into the multiple mechanisms of action of antimicrobial peptides, the impact of nonacylated and N-acylated hLFcin derivatives on morphology and cell division of E. coli was investigated and correlated with their effects on the phospholipids PE and CL. The results indicate that N-acylated peptides act via interaction with these lipids by a different mechanism than their nonacylated parent peptide. The former elicits a stronger perturbation of membrane lipid organization and also inhibits the cell division process.


To visualize the dynamics of the mannose 6-phosphate receptors in living cells, we stably expressed in HeLa cells a fusion protein made of GFP fused to the transmembrane and cytoplasmic domains of the Man-6-P/IGF II receptor (or cation-independent mannose 6-phosphate receptor, CI-MPR). A representative clone was selected for further studies. Pulse-chase experiments indicated that the level of expression of GFP-CI-MPR was ≈3–4 times higher than that of the endogenous CI-MPR in these cells and that the two proteins had similar half-lives (unpublished data). However, the expression of the GFP-CI-MPR did not significantly affect the trafficking of the endogenous MPRs because no significant missorting of lysosomal enzymes could be detected (unpublished data). Finally, antibody uptake experiments indicated that similar amounts (up to 30% within 2 h) of GFP-CI-MPR and endogenous CI-MPR passed through the cell surface and recycled back to the TGN indicating that both proteins traffic in a similar manner (unpublished data).

Cellular Distribution of the GFP-CI-MPR

A first examination of GFP-CI-MPR–expressing HeLa cells showed that this fusion protein distributed to different intracellular compartments (Figure A). The bulk of the fluorescent protein (≈90%) was present in the perinuclear region where the TGN and the late endosomes are usually located. Significant amounts of GFP-CI-MPR were also detected in small tubular structures scattered throughout the cytoplasm. This fluorescence pattern was not changed after cycloheximide treatment, indicating that these small tubular structures were different from the endoplasmic reticulum (unpublished data). Using transient expression, we first introduced different TGN markers in these cells, namely the VSVG-tagged sialyltransferase (Rabouille et al., 1995) and the gpI envelope glycoprotein of the varicella-zoster virus, a transmembrane protein that cycles between the TGN, the plasma membrane, and the endosomes (Alconada et al., 1996). The bulk of GFP-CI-MPR present in the perinuclear region almost completely colocalized with sialyltransferase, gpI, and the endogenous CI-MPR at the fluorescence level (Figure , B–D). Interestingly, the GFP-CI-MPR labeled long tubular processes protruding from this perinuclear compartment in several cells. If these tubular elements could also be decorated with anti-gpI or anti–CI-MPR antibodies, they appeared to exclude the VSVG-tagged sialyltransferase. Thawed cryosections of HeLa cells expressing the GFP-CI-MPR and the VSVG-tagged sialyltransferase were also labeled with a polyclonal antibody against GFP and a mAb against the VSVG-epitope followed by colloidal gold. Electron microscopy (Figure F) shows that the bulk of the GFP-CI-MPR was found in tubular vesicular structures reminiscent of the TGN, located in the close vicinity of the Golgi stacks containing the sialyltransferase. No significant labeling was detected over late endocytic compartments (unpublished data). We also internalized fluorescently labeled transferrin for 10 min at 37°C to identify early endocytic compartments. The internalized transferrin could be detected in ≈30% of the peripheral, GFP-labeled structures (Figure E). Altogether, these data indicate that the bulk of the GFP-CI-MPR localizes to the TGN and that small but significant amounts are present in early endosomes containing endocytosed transferrin as well as in peripheral structures devoid of this marker.

Distribution of the GFP-CI-MPR chimeric protein. (A) HeLa cells stably expressing GFP-CI-MPR were fixed and examined by fluorescence microscopy. (B and C) HeLa cells stably expressing GFP-CI-MPR (green) and expressing transiently markers of the trans-Golgi network, namely gpI (B) or a VSVG epitope-tagged sialyltransferase (ST C). The cells were fixed and immunolabeled with a mAb against the VSVG epitope or the SG1 anti-gpI mAb followed by a Texas Red–conjugated goat anti-mouse antibody (red). (D) HeLa cells expressing GFP-CI-MPR (green) were fixed and labeled with a polyclonal antibody against the luminal domain of CI-MPR (red). (E) These cells were also allowed to internalize Alexa594-labeled transferrin for 10 min at 37°C (Tf-Alexa594 E). (B–D) The GFP signal in the Golgi region (E) GFP signal in the periphery of the cell. Merged images are presented in the right column. Arrows indicate long tubules emanating from the perinuclear region. Arrowheads in E indicate examples of the overlap between both signals. Bars, 10 μm. (F) Thawed thin sections of HeLa cells stably expressing GFP-CI-MPR and transiently expressing the VSVG epitope-tagged sialyltransferase were double immunolabeled with a polyclonal anti-GFP antibody (5 nm colloidal gold, arrow) and a monoclonal anti-VSVG antibody (10 nm colloidal gold). Bar, 0.2 μm.

Dynamics of GFP-CI-MPR in the TGN

Using time-lapse videomicroscopy, we then visualized the dynamics of the GFP-CI-MPR located in the TGN. Figures A and 4A illustrates that the GFP-CI-MPR is sorted from the TGN in tubular structures of various lengths. These tubules are highly dynamic elements. With time, they elongated with an average speed of ≈0.9 μm/s, sometimes forming branched structures at the tip. Smaller tubular fragments could detach from the growing tubules and moved toward the cell periphery with an average speed of ≈0.9 μm/s (Figure B). These long tubular processes, which occasionally reached a length of 10 μm, could detach from the TGN and break into several smaller tubular fragments that moved toward the cell periphery with an average speed of ≈0.9 μm/s, most likely along microtubules. The statistical analysis of the dynamic state of these TGN-connected tubules (Table ) shows that ≈4 tubular elements with an average length of 6 μm could form within 2 min. Tubule formation, frequently occurring on the same restricted domains of the TGN, usually takes place with an average duration time of 20 s.

Formation of tubular elements from the TGN. (A) HeLa cells expressing the GFP-CI-MPR were examined by videomicroscopy. Images were taken at 2-s time intervals. Inverted images at the indicated time intervals are displayed. Arrowheads or squares indicate the tips of TGN-tubules, the arrows indicate the detachment of long tubular elements from the TGN and the breaking points into smaller tubular elements. The short line indicates a more intense GFP labeling that moves along the tubule toward the tip and then detaches from it. (B) Movement of TGN-derived tubules toward the cell periphery. Images were collected at 1-s intervals for 1 min. Paths of different TGN-derived tubules were traced as colored lines. The circles indicate fusion with GFP-CI-MPR containing structures. Bars, 10 μm.

TGN-tubules in GFP-CI-MPR-expressing cells were analyzed under the conditions indicated (for more details, see MATERIALS AND METHODS and RESULTS). Values correspond to means ± standard deviation. n in the columns “Number of tubules” and “Speed” indicates the number of cells and growing events of tubules, respectively, while n in “Length” and “Range of duration” columns indicates the number of tubules observed. The values marked with an asterisk are underestimated due to limitations of the time and frame sizes in the different recording conditions. n.d., tubules were not detected.

The dynamic state of the tubular elements depends on several factors (Table ). First, these events are temperature dependent. When GFP-CI-MPR–expressing cells were maintained at 20°C, a temperature known to drastically reduce protein sorting in the TGN, only few tubular elements (≈1 per 2 min) with a shorter length (≈2.8 μm) were seen. Under those conditions, the speed of elongation was also decreased (0.5 μm/s). Second, tubule formation requires the presence of cytoskeleton elements. This could be monitored when GFP-CI-MPR–expressing HeLa cells were treated with nocodazole, a drug known to destabilize microtubules. In nocodazole-treated cells, the Golgi rapidly fragmented into smaller elements scattered in the cytoplasm as previously described (Scheel et al., 1990). However, AP-1 and clathrin remained in a large part associated to these scattered elements (unpublished data). GFP-labeled tubules were unable to grow from these scattered Golgi elements. In a similar manner, tubule formation was almost completely abolished when cells were pretreated with cytochalasin D, a drug destabilizing the actin network. Cytochalasin D did not affect the distribution of coat components such as AP-1 and clathrin (unpublished data). Altogether, these results show that tubule formation depends on the temperature, requires the presence of both an intact microtubule network and actin filaments.

The ARF-1 GTPase, but not Wortmannin-sensitive PI-3 kinases, Regulates TGN-derived Tubule Formation

Several studies have now shown that the ARF-1 GTPase regulates the translocation onto membranes of AP-1 and GGAs, two coat components involved in MPR trafficking (Stamnes and Rothman, 1993 Traub et al., 1993 Le Borgne et al., 1996 Zhu et al., 1998 Meyer et al., 2000 Nielsen et al., 2001 Puertollano et al., 2001a Takatsu et al., 2001 Zhu et al., 2001). We therefore expressed ARF-1Q71L, a mutant impaired in GTP hydrolysis, in GFP-CI-MPR–expressing HeLa cells using a vaccinia recombinant virus. Under those conditions, the Golgi appeared as a cluster of smaller, GFP-labeled elements, still concentrated in the perinuclear region (unpublished data). As illustrated in Table , no tubule could form from these GFP-labeled compartments, indicating that GTP hydrolysis by ARF-1 regulates tubule formation.

In contrast, brefeldin A (BFA), which blocks the translocation of ARF-1 on membranes (Klausner et al., 1992) and the subsequent binding of AP-1 (Robinson and Kreis, 1992 Wong and Brodsky, 1992) and GGAs (Boman et al., 2000 Dell'Angelica et al., 2000 Hirst et al., 2000), results in the formation of a tubular network containing both TGN and endosomal markers (Lippincott-Schwartz et al., 1991). We then incubated the GFP-CI-MPR–expressing HeLa cells with BFA. The dynamics of tubules emanating from the TGN rapidly changed within 1–3 min after addition of BFA (Figure ), and after 10 min, BFA treatment resulted in the formation of a tubular network containing both the GFP-CI-MPR and the transferrin receptor (unpublished data). The statistical analyses shown in Table indicate that the number of tubules growing from the TGN with an average speed of 0.9 μm/s was largely increased in BFA-treated cells. On average, BFA-treated cells could form three times more tubules than untreated cells. Furthermore, these BFA-induced tubules were more stable (duration time of 96 s) and longer (average length of 14 μm) than in untreated cells because they could not break into smaller elements. These long tubules also exhibited a tendency to form highly dynamic branched structures at their tips. The fluorescence intensity of these tubules usually increased with time, probably reflecting the free diffusion of the GFP-CI-MPR in these tubular structures, which otherwise would have been kept retained in the TGN. As expected from previous studies (Lippincott-Schwartz et al., 1990), the BFA-induced formation of tubules was prevented by the addition of nocodazole, the microtubule-disrupting agent (unpublished data). Thus, the treatment of cells with BFA results in an enhanced formation and in a higher stability of tubular elements growing from the TGN. Altogether, these results indicates that the formation of TGN-derived tubules is controlled by the ARF-1 GTPase. By contrast, the treatment of GFP-CI-MPR–expressing cells with wortmannin, an inhibitor of PI-3-kinases shown to affect lysosomal enzyme sorting in mammalian cells (Brown et al., 1995 Davidson, 1995), did not significantly modify the dynamics of the TGN-derived tubular elements (Table ). Thus, the growth of TGN-derived tubules is probably not regulated by wortmannin-sensitive PI-3 kinases.

Effect of brefeldin A. (A) Images of GFP-CI-MPR–expressing cells were taken at 2-s time intervals during BFA treatment. Inverted images at the indicated time intervals (min:sec) are presented. The first frame (0:00) shows the cell immediately after adding BFA. Bar, 10 μm. (B) Growth of tubular elements in the absence (BFA−) or the presence (BFA+) of brefeldin A. Four examples of growing TGN tubules are shown.

Fate of the TGN-derived Tubules

The time-lapse sequences illustrate the saltatory movement of fluorescently labeled TGN-derived tubular elements along microtubules toward the cell periphery (Figures and ). However, some of these detached tubular processes sometimes reversed directions, suggesting that they could occasionally fuse back with the TGN. Therefore, we examined in more detail the fate of these GFP-labeled tubules emanating from the TGN. Figure shows a video sequence taken at three frames/s of a tubular element detaching from the TGN, moving toward the cell periphery, remaining stationary for a few seconds and then mixing with other small peripheral structures also labeled with the GFP-CI-MPR. The resulting structure was also very dynamic. It rapidly fragmented into two smaller elements that fused back again. Finally, the resulting GFP-labeled structure underwent fragmentation to give rise to two distinct structures (one vesicular and another more tubular) moving toward different directions. These peripheral structures labeled with GFP are also very dynamic elements. The fast recording presented in Figure B shows that they could make contacts between each other, probably reflecting fusion events, before fragmenting into separate structures. This observation could support the notion that they form a dynamic network continuously fusing and breaking in order to mix/exchange their content. We then asked whether these peripheral, GFP-labeled structures were dynamically connected with endocytic compartments. To visualize this, HeLa cells were allowed to internalize for 10–15 min Alexa594-transferrin bound to the cell surface. Figure C shows GFP-CI-MPR-containing tubular elements and transferrin-positive structures establishing tight contacts, giving rise to structures in which the two fluorescent markers overlap. Within a few seconds, the two markers segregate again, each being packaged into separate structures. Thus, these data strongly suggest that the TGN-detached tubules can fuse with peripheral GFP-CI-MPR– containing structures and that these latter structures can exchange their content with endocytic compartments containing internalized transferrin.

Fate of tubular elements detaching from the TGN. (A) TGN-derived tubular elements mix with GFP-labeled peripheral structures. An area of a GFP-CI-MPR–expressing HeLa cell was selected as indicated in the left panel and examined by videomicroscopy. Images were taken at a high speed (3 frames per second). Inverted images are presented. Sequences of images taken at the indicated time intervals are displayed in B. The arrow indicates a tubular element detaching from a TGN tubule and fusing with a peripheral compartment labeled with the GFP. (B) GFP-labeled, peripheral structures are highly dynamic. Cells were examined as in A. The arrow and the asterisk indicate different peripheral structures containing the GFP-CI-MPR. (C) Mixing of GFP-CI-MPR–positive structures with endocytic compartments. GFP-CI-MPR–expressing cells were incubated at 4°C with Alexa594-transferrin for 30 min and then washed and reincubated at 37°C for 10–15 min. The cells were examined by confocal microscopy. Images were recorded every second. The arrow indicates the overlap between the two fluorescent markers. Bar, 2 μm.

Dynamics of AP-1 Coats

The results described above prompted us to investigate whether the GFP-labeled tubular elements forming at the TGN could contain the machinery required for MPR sorting, in particular the AP-1 coat whose function in MPR trafficking remains unclear at present.

GFP-CI-MPR–expressing cells were first labeled with antibodies against the γ-subunit of AP-1 or against clathrin and examined by confocal microscopy. Figure shows that many GFP-labeled tubules could be decorated with anti–AP-1 and anticlathrin antibodies. It is worth noting however that AP-1 as well as clathrin do not distribute uniformly over the tubules but are detected on domains that appear to contain higher amounts of GFP-CI-MPR. As expected, AP-1 was also detected on the TGN as well as on several of the peripheral structures containing the GFP-CI-MPR (Figure , A and C). Second, a CFP-CI-MPR and a γ-subunit of AP-1 fused to YFP were coexpressed in HeLa cells. This γ-subunit of AP-1 fused to YFP was incorporated into a complex (unpublished data) able to bind to membranes in vivo (Figure A).

Distribution of endogenous coat components. (A–C) HeLa cells expressing GFP-CI-MPR (green) were fixed, labeled with either the 100.3 mAb against γ-adaptin (red AP-1 A and C) or a polyclonal antibody against clathrin (red B), and examined by laser confocal microscopy. (A and B) the signals in the Golgi region (C) signals in the periphery of the cell. Note that AP-1 or clathrin signals appear as concentrated spots along the TGN tubules (arrowheads).

Steady state distribution of YFP-tagged γ-adaptin. (A and B) HeLa cells expressing both CFP-CI-MPR (green) and YFP-γ-adaptin (red YFP-AP1) were treated without (A) or with BFA (B) for 10 min and fixed. (C) Hela cells expressing YFP-γ-adaptin (green) were also labeled with antibodies against clathrin (red). (D) HeLa cells expressing GFP-γ-adaptin (green GFP-AP1) were allowed to internalize Alexa594-labeled transferrin for 10 min at 37°C (red Tf-Alexa594). Merged images are presented in the middle column. Bars, 10 μm

Western blotting experiments (unpublished data) indicated that the YFP-tagged AP-1 was distributed between a cytosolic pool (60% of total) and a membrane-bound pool (40% of total) as the endogenous AP-1. The YFP-AP-1 bound to membranes became soluble upon BFA-treatment (Figure B). Overall, this YFP-AP-1 exhibited a similar distribution over the TGN and peripheral, tubular structures containing internalized transferrin, as the endogenous AP-1, and was detected in membrane domains also coated with clathrin (Figure C). Altogether, the data strongly suggest that the YFP tag on AP-1 γ-subunit does not affect the membrane binding properties of AP-1 or its interaction with clathrin. HeLa cells coexpressing CFP-CI-MPR and YFP-AP-1 were then examined using time-lapse confocal microscopy to investigate the dynamics of both AP-1 and CI-MPR in living cells.

Several types of events could be seen as illustrated in Figure . First, small tubular elements coated with YFP-AP-1 and containing the CFP-CI-MPR detached from the TGN and moved toward the cell periphery. This is in good agreement with the results reported by Sorkin and coworkers (Huang et al., 2001). Frequently, the YFP-AP-1 coat did not seem to be uniformly distributed along these tubular elements but appeared as patches moving along CFP-CI-MPR–labeled tubules as seen for the endogenous AP-1 in fixed cells (Figure A). Occasionally, TGN-derived tubules containing the CFP-CI-MPR moving toward the cell periphery appeared to lose their YFP-AP-1 coat. In other cases however, this coat remained associated with these structures, which could nevertheless fuse with peripheral tubular structures. As reported earlier, the YFP-AP-1 coated, CFP-CI-MPR–containing peripheral tubular structures appeared as highly dynamic structures. Although they were partly coated with YFP-AP-1, these structures could ultimately fuse with each other. It should be noted, however, that some tubular elements forming at the TGN appeared to be devoid of the YFP-AP-1 complex. The quantification indicates that 35% of the CFP-CI-MPR–containing tubules are devoid of YFP-AP-1 when they detach from the TGN. As shown in Figure , some of these tubules could acquire an YFP-AP-1 coat while they move toward the cell periphery. Altogether, these data show that AP-1 is present on structures where sorting of CI-MPR occurs, i.e., TGN and peripheral elements, probably endosomes as well as on transport intermediates carrying the CI-MPR.

Dynamics of YFP-AP-1 and CFP-CI-MPR. HeLa cells expressing both CFP-CI-MPR (green) and YFP-γ-adaptin (red YFP-AP1) were observed by time-lapse laser scanning microscopy. Images were taken every 1.5 s with multitrack mode. A selected area indicated in A is displayed in B and C at the time intervals as indicated. Merged images are shown at the bottom. (B) A TGN-derived tubular element (arrow) without any AP1-coat moves toward the periphery and acquires an AP1-coat. (C) Another tubular element with an AP1-coat (arrow) detaches from the TGN and appears to fuse with the peripheral compartment (asterisk) that has been formed in B. Bars, 10 μm.

Materials and Methods

Virus Detection, DNA Isolation, and Cloning.

Virus particles were purified by cesium chloride gradient to obtain viral DNA for cloning (detailed later) from the pooled, ∼500-g P. monodon-infected tissue, obtained from Vietnam. After homogenization in PBS and initial extraction with chloroform/butanol (1:1 volume), a clear supernatant containing viral particles was obtained by low-speed centrifugation. Virus stock was concentrated from the supernatant by ultracentrifugation at 40,000 rpm in a type 60Ti rotor for 2 h at 4 °C. Pellets were resuspended in small volume of PBS for DNA extraction or EM analysis. Viral DNA was extracted by the High Pure Viral Nucleic Acid Kit (Roche) and eluted in 40 μM of distilled water. The isolated DNA was blunt-ended utilizing T4 DNA polymerase and Large Klenow fragment of DNA polymerase I (New England Biolabs) in the presence of 33 μM of each dNTP and cloned into the EcoRV restriction site of a pBluescript KS + vector. Starting with the M13 primer sites of the vector, the sequence of the PmMDV genome could be determined by primer walking. To obtain the sequences of the termini, several GC-rich cutter restriction enzymes were used to release secondary structures and the fragments subcloned and sequenced. Two GC cutters proved to be sufficient, namely ApaI and HaeIII, enabling cloning into the ApaI and EcoRV sites of the pBluescript KS − and KS + vectors, respectively. We used the Sure Escherichia coli strain (Stratagene), and incubation at 30 °C, for both infectious clone construction and for subcloning of viral ITRs.

Cell Lines, Transfection, and Culturing Conditions.

Sf9 (ATCC CRL-1711), C6/36 (ATCC CRL-1660), and Schneider’s Drosophila Line 2 (ATCC CRL-1963) were tested for PmMDV susceptibility however, none of these could sustain PmMDV replication. To perform transcription and expression studies, the Bac-to-Bac expression system was used (Invitrogen, Thermo Fisher Scientific), involving the transfection of Sf9 cells. Sf9 cultures were maintained in SF900 II medium (Gibco) in a serum-free system at 28 °C. Cellfectin II Reagent (Invitrogen) was used for DNA transfection with 8 × 10 5 cells per well, previously seeded on a six-well culturing dish and incubated overnight at 28 °C. The culturing medium was aspired and replaced by seeding medium of Grace’s complete insect medium supplemented with 5% FBS (Gibco) and Graces’s unsupplemented insect medium, mixed at a ratio of 1:6, respectively. After adding the transfection reagent–DNA mixture to the wells, cells were incubated for 5 h. The aspired transfection medium was replaced with SF900 II medium supplemented with 2% FBS. Cells were checked daily for signs of cytopathic effects (CPE) and the whole culture was collected when 70% of the cells detached from the dish or showed granulation. This was followed by three cycles of freeze–thaws on dry ice and 200 μL of this passage 1 (P1) stock was transferred to 25 mL of fresh Sf9 suspended cell culture in polycarbonate Erlenmeyer flasks (Corning) at the density of 2.5 × 10 6 cells/mL, to create the P2 stock, cultured in serum-free SF900 II medium without antibiotics.

Transcription Studies.

To study the transcriptome of PmMDV, we cloned the entire viral genome, including the ITRs, into a pFB dual vector, where both the p10 and the PH (polyhedrin) promoters had been knocked out previously hence, transcription of the whole PmMDV genome could be analyzed in the recombinant Autographa californica nuclear polyhedrosis virus (NPV) genome without interference of the NPV promoters. This construct was designated PmMDV-Bac-complete. The yield of DNA, however, of bacmid minipreps from the DH10Bac (Invitrogen) cells was below 500 μg/mL hence, the large ITRs were removed. This yielded over 2 mg/mL bacmid DNA of each preparation. Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research), where the denaturation step was executed by adding TRIzol Reagent (Thermo Fisher Scientific). RNA was treated by digestion with the TURBO DNA-free Kit (Ambion) to get rid of residual DNA contamination, as well as subjected to a control PCR for the remaining DNA fragments. Reverse transcription was performed only on entirely DNA-negative preparations using the SuperScript IV or the SuperScript III enzymes (Thermo Fisher Scientific), supplemented with random nonamers (Sigma-Aldrich). To avoid false-detection of splicing, isolated and Dnase-treated RNA was dephosphorylated by adding Antarctic phosphatase (New England Biolabs) and incubated for 30 min at 37 °C. Primers were designed at the following positions of the PmMDV genome: 1,100 nt (reverse and forward), 2,300 nt (reverse and forward), 2,564 nt (reverse and forward), 3,038 nt (reverse), and 3,511 (reverse and forward).

Anchored oligo(dT) primers were used together with the 2,300 forward and 3,511 forward primers for 3′ RACE (rapid amplification of cDNA ends). To perform 5′ RACE to map transcription start sites, we designed adaptors with the sequence of ATC​CAC​AAC​AAC​TCT​CCT​CCT​C’3. Dnase-treated RNA was subjected to dephosphorylation. by alkaline calf intestinal phosphatase (New England Biolabs) and after phenol-chloroform extraction to dephosphorization by tobacco acid pyrophosphatase (Ambion) to remove 5′ RNA caps. After the ligation of adaptors using T4 RNA ligase (New England Biolabs), reverse transcription was executed as described above. PCR was performed with the readaptor primers together with oligos 1,100 reverse and 2,564 reverse. All PCRs were performed using Phusion Hot Start Flex DNA Polymerase (New England Biolabs) in a 25-μL final reaction volume, including 2 μL of purified cDNA target, 0.5 μL of both primers in 50-pmol concentration, 0.5 μL dNTP mix with 8 μmol of each nucleotide, 0.75 μL of 50 mM MgCl2 solution, and 0.25 μL of enzyme. PCR reactions were executed under a program of 5-min denaturation at 95 °C followed by 35 cycles of 30-s denaturation at 95 °C, 30-s annealing at 48 °C, and 1 or 2 min of elongation at 72 °C. The final elongation step was 8-min long at 72 °C. In case of the 5′RACE reactions, 0.5 μL of enzyme was used and the number of cycles was reduced to 25. For the 3′RACE, the reaction was supplemented with 1 μL of 50 mM MgCl2 and the annealing step was left out.

Protein Expression and Purification of VLPs.

The plasmids PmMDV-bac-complete and PmMDV-Bac-p47 were constructed by using a pFB dual vector (Invitrogen), from which both the polyhedrin (PH) and the p10 promoters had been removed, while PmMDV-Bac-ORF4 was of pFB1 backbone, driven by the PH promoter (Invitrogen). For the expression studies, the P2 baculovirus stock was used in the case of all three constructs detailed above. The P2 stocks were incubated for at least 7 d and monitored for CPE every third day. When at least 70% of the cells showed signs of CPE, the culture was collected, centrifuged at 3,000 × g, and the pelleted cells disrupted by three cycles of freeze–thaws on dry ice. This lysed cell pellet was then resuspended in 1 mL of 1× TNTM pH8 (50 mM Tris pH8, 100 mM NaCl, 0.2% Triton X-100, 2 mM MgCl2) and centrifuged again. Supernatant was mixed back together with the cell culture supernatant and was subjected to treatment with 250 units of Benzonase Nuclease (Sigma-Aldrich) per every 10 mL. The liquid was mixed with 1× TNET pH8 (50 mM Tris pH8, 100 mM NaCl, 0.2% Triton X-100, 1 mM EDTA) in a 1:1 ratio and concentrated on a cushion of 20% sucrose in TNET, using a type 60 Ti rotor for 3 h at 4 °C at 45,000 rpm on a Beckman Coulter S class ultracentrifuge. The pellet was resuspended in 1 mL of 1× TNTM pH8 and after overnight incubation purified on a 5 to 40% sucrose step gradient for 3 h at 4 °C at 35,000 rpm on the same instrument in a SW 41 Ti swinging bucket preparative ultracentrifuge rotor. The visible single band that formed at the 15 to 20% sucrose interface was then collected by needle puncture and a 10-mL volume syringe. In the case of constructs PmMDV-Bac-complete and PmMDV-Bac-p47, both expressed by the own promoters of PmMDV, sucrose gradient purification did not result in a visible band hence, the cushion-concentrated pellet after TNTM resuspension was purified in cesium chloride instead, dissolved in TNTM at a density of 1.38 g/cm 3 . After 24-h ultracentrifugation at 40,000 rpm at 16 °C in a SW 55 Ti rotor, the VLPs could be aspired using a needle and a 1-mL syringe. The aspirate was dialyzed into 1× HCB buffer (50 mM Hepes, 4.3 mM MgCl2 × 6H2O, 0.15 M NaCl) at pH 7.4 to remove the sucrose or the cesium chloride. As PmMDV demonstrated better stability at the higher pH of its natural extracellular environment, particles purified were dialyzed into 1× universal buffer (20 mM Hepes, 20 mM MES, 20 mM sodium acetate, 0.15 M NaCl, 3.7 mM CaCl2) at pH 8.2, which is equivalent with the pH of tropical marine water.

In Silico Analyses.

After obtaining the sequence of the ITRs and genome clones, the complete genome sequence of PmMDV was assembled using Staden package v4.11.2 (69). The assembled genome was annotated, as well as the transcripts assembled and splice sites investigated in Artemis Genome Browser by the Sanger Institute (70). Homology searching at amino acid levels was carried out two ways: To determine sequential similarity, the Blast algorithms were applied with different expectation value levels (71), whereas structural similarity was predicted by the genThreader, pDomThreader, and pGenThreader algorithms of the PSIpred web server ( (72). To investigate the conserved motifs and domains with known homologs in the derived aa sequences, the DomPred algorithm of the PSIpred server as well as the SMART web application was used (73). Structural similarity of the resolved capsid structures with those available in the RCSB Protein Data Bank (PDB) was investigated using the DALI server (74). The tBLASTn algorithm was utilized to screen the RefSeq, Whole Genome Shotgun Contigs, High-Throughput Genomic Sequences, and Transcript Shotgun Assembly databases, targeting 5,000 hits with an expectation value of 10 (71).

For phylogenic inference, alignments, incorporating the outputs of pairwise, multiple, and structural aligners, were constructed using the Expresso algorithm of T-Coffee (75). Structural data were obtained using the PDB mode of this algorithm. The constructed alignment was further edited using Unipro Ugene (76). Model selection was executed by ProtTest v2.4, suggesting the LG + I + G + F substitution model based on both the Bayesian and Akaike information criteria (77). The distance matrix to the starting trees were constructed using the Prodist program of Phylip v3.695 with a Johns–Thorton–Taylor method and the starting tree was constructed from this using the Fitch–Margoliash method of the Fitch program with global rearrangements (78). Bayesian inference was executed by the BEAST v1.10.4 package, incorporating the predicted LG + I + G + F model, using a log-normal relaxed clock with a Yule speciation prior, throughout 50,000,000 generations (79). Convergence diagnostics were carried out using Tracer v1.7.1, which indicated the Markov-chain Monte Carlo runs to have converged (80). Phylograms were edited and displayed in the FigTree 1.4.1 program of the Beast package.

To investigate possible homologous VPs throughout the family, we used the Blast P and PSI Blast NCBI algorithms with an expect threshold of 1,000 targeting the maximum number of 5,000 sequences (81). As query, one VP-derived amino acid sequence was submitted for each recognized parvovirus species as well as one for each complete, unclassified entry. In the case of the PLA2-containing VPs, the N-terminal sequence, containing this conserved domain, was removed to avoid false hits. Screening was performed using the substitution matrices Blosum62 and PAM250. Two sequences were marked as homologs in case the search resulted in a hit. The search was limited to family Parvoviridae in order to filter out false positives.

To construct the model of the eight stranded β-barrel jellyroll core, which could be docked into the PmMDV high-resolution structure density, the I-TASSER Standalone Package v5.1 was used (82). As a template search failed to detect structural similarity with any PV capsid structure, threading was restricted to the PstDV VP monomer (PDB ID code 3N7X) and incorporated secondary structure predictions, obtained by the PSIpred server (72). To achieve the best possible fit of the core, the N- and C-terminal regions up to and following the first and last β-sheets were removed from the amino acid sequence.

Structural Studies.

The statistics for each reconstruction and refinement are given in SI Appendix, Table S3. Three-microliter aliquots of the PmMDV VLPs without/with EDTA (∼1 mg/mL) were applied to glow-discharged C-flat holey carbon grids with a thin layer of carbon (Protochips) and vitrified using a Vitrobot Mark IV (FEI) at 95% humidity and 4 °C. The quality and suitability of the grids for cryo-EM data collection was determined by screening with a 16-megapixel charge-coupled device camera (Gatan) in a Tecnai G2 F20-TWIN transmission electron microscope operated at 200 kV under low-dose exposure (∼20 e−/Å 2 ) prior to data collection. For collecting the low-resolution native and EDTA-treated PmMDV datasets, the same microscope was used at 50 frames per 10 s using a K2 direct electron detector (DED) at the University of Florida Interdisciplinary Center for Biotechnology Research electron microscopy core.

High-resolution data collection was carried out at two locations: the Florida State University (FSU) for the EDTA-treated PmMDV dataset, and the University of California, Los Angeles (UCLA) for the native dataset. At both locations, a Titan Krios electron microscope (FEI) was used, operating at 300 kV, equipped with a DE64 DED (Direct Electron Detector) at FSU and Gatan K2 DED at UCLA. At UCLA, the scope also contained a Gatan postcolumn imaging filter (Gatan) and a free-path slit width of 20 eV. Movie frames were recorded using the Leginon semiautomated application at both sites (83). At FSU, the frame rate was 50 per 10 s with ∼60 e−/Å 2 electron dosage. At UCLA, images were collected at 50 frames per 10 s with an ∼75 e−/Å 2 electron dosage. Movie frames collected at both locations, as well as for the low-resolution data set collected at the University of Florida, were aligned using the MotionCor2 application with dose weighting (84).

Systems Biology of Bacteria

Andreas Otto , . Dörte Becher , in Methods in Microbiology , 2012

1.2 Gel-based proteomics

Gel-based studies rely on two-dimensional gel electrophoresis (2DE) for the resolution of complex protein mixtures according to the pI and molecular weight (MW) of the individual proteins in a sample ( Neidhardt, 2011 ). A major asset of gel-based proteomics is the analysis of intact protein species providing direct access to post-translational modification (PTM) events, for example, changes in MW due to proteolytic processing/degradation or direct staining of phosphorylation events by ProQ Diamond ( Hecker et al., 2008, 2009 Rabilloud et al., 2009 ). Differential gel image analysis of 2D gels representing different proteomic snapshots delivers relative quantitative information solely based on the staining intensities of the protein spots found on the gels. Since the invention of 2DE, this technique has matured to become an exceedingly robust and relatively cheap technique in terms of equipment needed ( Westermeier and Marouga, 2005 Rabilloud et al., 2010 ). In microbiology, 2D gel-based proteomics is commonly used for assessment of adaptation responses to nutrient shifts ( Bernhardt et al., 1999 ), antimicrobial agents ( Wenzel and Bandow, 2011 ) or environmental stresses and starvation ( Budde et al., 2006 ). The advantages of 2D gels for the study of microbial physiology, namely, the visualization of the majority of the proteins involved in biosynthetic pathways and the main metabolic routes, have recently been reviewed for Gram-positive bacteria ( Völker and Hecker, 2005 Hecker et al., 2008 ). Elucidating changes in the amounts of key effectors in metabolism and cellular structure provides valuable insights into the main processes of life and is thus essential for systems biology approaches.

Lipase activity

In order to understand HDL metabolism, it is important to be aware that lipoproteins of each class are heterogeneous particles that heavily interact with, and often change into one another. A common factor influencing lipoprotein interaction is activity of lipase proteins. Lipases are water-soluble enzymes that hydrolyze ester bonds of water-insoluble substrates such as triglycerides, phospholipids, and cholesteryl esters (32). Endothelial lipase (LIPG), hepatic lipase (LIPC), and lipoprotein lipase (LPL) are three vascular lipase proteins that migrate to endothelial cells and anchor to the distal side via interaction with heparin sulfate proteoglycans (33-35). The exposed lipase proteins remodel circulating lipoproteins, generating important effects to lipoprotein metabolism and cholesterol homeostasis. Additionally, some lipase enzymes directly interact with lipoprotein receptors, such as the LDL receptor, enhancing metabolism of circulating lipoproteins.

The majority of the catalytic activity of LIPG is devoted to hydrolysis of phospholipids of VLDL, chylomicrons, and HDL (33). Interestingly, LIPG mediated phospholipid modulation of HDL inhibits cholesterol efflux from SR-BI, but enhances efflux from ABCA1, and HDL uptake in the liver (36). In 2006, Badellino and colleagues (37) established a correlation between LIPG and atherosclerosis, which underscores the importance of SR-BI cholesterol efflux and HDL longevity in ideal HDL function.

The LIPC protein is synthesized primarily by hepatocytes, and then secreted and bound to the extracellular matrix of hepatic endothelial cells (38). LIPC has powerful VLDL and IDL triglyceride hydrolysis capabilities (39), as well as the ability to catalyze conversion of α-HDL subspecies HDL2 to denser HDL3 (40) (Figure 1). The latter functionality has direct implications to RCT because HDL2 is more likely to interact with SR-BI for cholesterol efflux or endocytosis (41). Independently of RCT, LIPC appears to demonstrate pro-atherogenic effects by increasing artery wall retention of VLDL, chylomicrons, and LDL (42). Although mutations in human LIPC are associated with variations in HDL concentration, the connection with coronary artery disease is controversial (43, 44).

LPL is a critical enzyme involved in hydrolysis of triglyceride rich lipoprotein particles in muscle, adipose, and macrophages, a process which generates free fatty acids and glycerol for energy metabolism and storage (45). Expression of LPL has been implicated in atherosclerosis, citing the increased affinity for macrophage phagocytosis (and subsequent foam cell development) on LDL and chylomicron particles after LPL mediated remodeling (46).


The coral reef is a highly competitive place, where the morphologically simple and sessile corals need to be able to “fight” in parallel on different fronts – catching prey, defending themselves against predators and competing in territorial aggressive encounters with other corals. We propose a conceptual model describing some physiological and molecular features of the two different tentacle types of Galaxea fascicularis, each of which has evolved to address a different ecological challenge. Our results suggest that the CTs are adapted to catch prey using paralytic toxins and hemolysins, likely injected through the MpM nematocytes, with spirocytes helping to entangle the prey. The prey is then transferred to the mouth using cilia combined with a mobile mucus layer, and the ciliary beating is controlled by the Histamine H2 system. The ST, in contrast, actively “search” for their long-distance targets, with the tentacle length and movement (and potentially the nematocyte discharge) controlled by the opsin and allostatin sensory systems. Once the target organism is identified, the ST deliver primarily enzymatic venom through the two different MbM nematocyte types, as well as (potentially) as part of the extensive non-nematocytic mucus secretion. We speculate that the ST mucus may have a higher relative composition of membrane-bound glycoproteins (such as mucins), thus being less of a “mobile” or flowing mucus cover. This mucus might also play a role in the recognition of self vs non-self tissue. This conceptual model is based on histological observations, functional toxicity assays and analyses of differential gene expression, and provides several hypotheses that can be tested using experimental tools currently available for non-model corals such as Galaxea fascicularis.

The venom of one organism – the coral - needs to produce three different functional outcomes – paralysis, pain and tissue degradation. The need for venom to fulfill different roles is not unique to corals – scorpions, cone snails and assassin bugs, for example, each produce multiple venom cocktails that nevertheless are injected through the same venom apparatus [25, 46, 90]. In the case of the coral, the morphological distinction between the CT and ST enable each to produce a unique chemical armament. However, it is also possible that within each tentacle type there may be more than one type of venom. For example, in Hydra, different nematocytes are discharged in response to prey and to predators [29, 84]. Additionally, many cnidarians also employ toxins that are not delivered through the stinging cells [19, 64, 83]. It remains to be tested whether, within each tentacle type, different effector molecules or toxins are delivered through different cell types. If so, this “division of labor” might enable a complex chemical armament to be delivered in a target- and time-specific manner.

Our results also leave many questions open. For example, the genes more abundantly expressed in the ST are enriched with the GO terms “oxygen binding” and “heme”, yet the relationship between these molecular functions and the biology of the STs is still unclear. Genes encoding RNAase, transcription factor binding and ubiquitin-protein transferase activities are also enriched in the ST, suggesting a constant dynamic remodeling of these tentacles, a process that has yet to be studied in detail. In contrast, despite the clear differences in cilia and flagella between the two tentacle types, no differences were observed in the expression of their structural genes. It is possible that post-translational regulation plays a role in the regulation of the development and function of these organelles. Finally, approximately 34% of the genes identified in the transcriptome could not be functionally annotated, including 4% of the genes differentially expressed between the CT and the ST. This highlights the richness of molecular functions still to be discovered that allow these corals to survive and thrive on the coral reef.


Nowadays, gene fusion techniques are indispensable tools in a variety of biochemical research areas. 32 Recombinant chimeric fusion proteins are routinely constructed to increase the expression of soluble proteins and to facilitate protein purification. 32 , 83 Other engineering approaches that link two proteins or protein domains by a peptide linker include immunoassays (e.g., using chimeras between antibody fragments and proteins 84 , 85 ), selection and production of antibodies, 86 and engineering of bifunctional enzymes. 87

In the respiratory chain, electron transfer protein domains of flavodoxin and cytochrome c553 from Desulfovibrio vulgaris and the heme domain of P450 BM3 from Bacillus megaterium have been used as molecular “Lego”-type building blocks in different combinations to build artificial redox chains having variable redox potentials. 88 Multidomain bacterial protein toxins have been used in designing potential carriers for targeted delivery of biomolecules. 89 Catalytically functional flavocytochrome chimeras 90 and modified cellular signaling circuits through modular recombination of domains 91 are some of the other recently reported chimeric proteins having additional functions introduced into them through engineered domains and linkers.

The selection of the linker sequence is particularly important for the construction of functional chimeric proteins. 32 A recent study on the streptococcal protein G-Vargula luciferase chimera suggested that the spatial separation of the heterofunctional domains of a chimeric protein by an appropriate linker peptide is important for the domains to work independently. 92 In a similar study, Arai et al. designed linkers to effectively separate the two domains of a chimeric protein. 83 The authors introduced helix-forming peptide linkers, (EAAAK)n, between two green fluorescent protein (GFP) variants. Circular dichroism (CD) spectroscopic analysis suggested that the introduced linkers form an α-helix, and that the α-helical contents increase as the lengths of the linkers increase. Fluorescence resonance energy transfer (FRET) observed between the two GFP variants also suggested that the distance between the two GFP domains increases as the lengths of the linkers' increase.

It is difficult to determine the orientation of the domains and the conformation of the linker of the chimeric proteins by FRET and CD analyses alone. Therefore, a direct visualization of the three-dimensional (3D) structures in solution is desirable. To gain information about the general shape, it is not necessary to solve structures at atomic resolution. Single particle cryo-EM routinely yields low-resolution (10–30 Å) shapes of particles larger than about 300 kDa molecular weight. 93 Synchrotron X-ray small-angle scattering (SAXS) has emerged in recent years as a complementary low-resolution alternative for the investigation of smaller chimeric proteins. Computational techniques 94-97 allow one to deduce the 3D shape from one-dimensional (1D) SAXS scattering profiles that correspond to the isotropically averaged reflections of X-rays on the randomly oriented particles in solution. In Ref. 32, the shapes and sizes of the chimeric proteins, consisting of GFP variants with the helical linkers (EAAAK)n (n = 2–5) and flexible linkers (GGGGS)n (n = 3, 4), were deduced from the SAXS diffraction pattern with an ab initio modeling procedure. Figure 5 shows two of the resulting models.

Modeling of atomic structures into 3D shapes from SAXS. 32 , 97 Variants of green and blue fluorescent protein 32 connected by (a) (EAAAK)4 and (b) (EAAAK)5 linkers (cartoon representation). The SAXS envelopes and fluorophores are shown in green. The β-sheets are shown in yellow and α-helices in blue.

The SAXS experiments demonstrated that short helical linkers (n = 2, 3) cause multimerization, while the longer linkers (n = 4, 5) solvate monomeric chimeric proteins. 32 Also, chimeric proteins with a helical linker assumed a more elongated conformation compared to those with a flexible linker. The elongation depends on the length of the helical linker element in agreement with the molecular ruler hypothesis (Absence of Flexibility section). The chimeric proteins with the flexible linker exhibited an elongated conformation as well, rather than the most compact side-by-side conformation expected from FRET analysis. Information about the global conformation of the chimeric protein is thus necessary for optimization of the linker design. 32

Linker engineering, with the aim to control the distance, orientation, and relative motion of two functional domains, will increase in importance with increasing emphasis on the de novo design of multidomain proteins. A number of recent databases and surveys aid in the design of linkers for chimeric proteins. 73 However, despite many empirical surveys, very little is known about the structural factors that govern interdomain flexibility. Such lack of knowledge is a limiting factor in de novo chimera design. Therefore, a number of recent studies focused on the structural principles governing the domain architecture and their assembly. 98-107 The emerging concepts, along with the bioinformatics tools that attempt to detect domains and their motions from sequence information alone, 108 may one day lead to a precise de novo engineering of interdomain flexibility, thereby helping achieve the desired functioning of synthetic chimeras. In the mean time, the successful use of feedback techniques such as CD, FRET, and SAXS suggests that gene fusion applications should be accompanied by geometric analysis for appropriate biophysical validation of the linker design process.

Watch the video: Visualization - Information Visualization III: Visualization Systems and Visualization Design (October 2022).