Thermoresponsive Chiral to Nonchiral Ordering Transformation in the

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Thermo-responsive chiral to non-chiral ordering transformation in the nematic liquid crystal phase of rodlike viruses: turning the survival strategy of a virus into valuable material properties Shuaiyu Liu, Tingting Zan, Si Chen, Xiaodong Pei, Henmin Li, and Zhenkun Zhang Langmuir, Just Accepted Manuscript • Publication Date (Web): 08 Jun 2015 Downloaded from http://pubs.acs.org on June 9, 2015

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Thermo-responsive chiral to non-chiral ordering transformation in the nematic liquid crystal phase of rodlike viruses: turning the survival strategy of a virus into valuable material properties Shuaiyu Liu1, Tingting Zan1,2, Si Chen1, Xiaodong Pei1, Henmin Li1, Zhenkun Zhang1,* 1. Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin, 300071, China; 2. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

E-mail: [email protected] KEYWORDS: chiral nematic liquid crystal phase • virus • coat protein •gold nanorod • reconfigurable self-assembly

ABSTRACT: The current work investigates the thermo-responsive in situ chiral to non-chiral ordering transformation of a rodlike virus in the naturally assembled state the chiral nematic liquid crystal (CLC) phase. We take this as an elegant example of reconfigurable self-assembly, through which it is possible to realize in situ transformation from one assembled state to another without disrupting the pre-formed assembly in general or going through a secondary assembling procedure of the disassembled building blocks. Detailed investigation presented here reveals many unique characteristics of the thermos-responsive 3D chiral ordering of rodlike viruses

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induced by heat stress. The chiral to non-chiral ordering transformation is highly reversible in the temperature range of up to 60 oC and can be repeated for many times. There exists a critical temperature around 40 oC which is independent of the ionic strength and virus concentration. Such reconfigurable ordering in the CLC phase stems from the intrinsic structure change of constituent coat proteins without disrupting the structure integrity of the virus, as revealed by three analytical techniques targeting levels ranging from the molecular, secondary conformation of the constituent proteins, to the whole single virus, respectively. Such structure flexibility, also termed as polymorphism, is relative to the survival stagey of a biological organism such as the virus and can be transformed into very precious material properties. The potential of the virus based CLC phase as the chiral matrix to regulate chiro-optical properties of gold nanorods is also presented.

1. Introduction With the explosive development of the synthesis methodology, nowadays it is possible to prepare various kinds of nano or colloidal particles made of a wide spectrum of materials and possessing rich shapes. The current challenge is to further assemble these particles into regular aggregates if the idea for functional materials via bottom-up self-assembly has to come into reality.1-2 In this regard, significant progresses have been made in the past decades, resulting in many strategies that can precisely guide the self-assembly of nano or colloidal particles into targeted assemblies with high fidelity and abundance.3-7 Normally, nano or colloidal particles self-assemble into a targeted assembled state or superstructure following certain way


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and then stay in that assembled state. It is highly desirable to realize in situ transformation from one assembled state to another without disrupting the pre-formed assembly in general or going through a secondary assembling procedure of the disassembled building blocks.6 Such envision has slowly evolved into a new concept reconfigurable self-assembly, i.e. a self-assembling method to reconfigure the relative special position or ordering of the constituent particles in the pre-formed self-assembled structure, often involving external stimuli.8-9 The advantage of reconfigurable self-assembly is obvious: many collective material properties of the assembled functional materials, such as optics, surface plasmon resonance and catalysis, etc., are related to the inter-particle distance and relative position of the constituent particles in the assembled structure.10-11 The implementation of reconfigurable self-assembly is expected to produce new intelligent, adaptive and actuating soft materials.12 However, examples of reconfigurable self-assembly, if not impossible, are definitely rare. Pioneering works have been demonstrated by Ganick and coworkers.13-14 For instance, they have created single ribbons and multiple rings self-assembled from Janus magnetic colloidal rods that can undergo in situ hierarchical reconfiguration into many other structures regulated by magnetic fields.14 Recently, a remarkable work form Dogic and coworkers has demonstrated a flat colloidal membrane can transform into many exotic morphologies through chiral control of interfacial tension.9 In situ transformation between assembled states can be realized by reordering, changing relative position or intrinsic properties of the building blocks inside the


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pre-formed assembled structure.13-15 The former two means have been realized through endowing the building blocks with magnetic properties, as exampled by several recent works.13-15 However, it is relatively challenging to realize reconfigurable self-assembly by tuning intrinsic properties of the building block, like flexibility, size and shape, surface properties, etc.16 In the case of organic or inorganic nanocolloidal particles, their shape, internal structure and surface properties are fixed during preparation and are usually not possible to be tuned. In contrast, many biological nanoobjects, like F-actins, flagella and viruses, have tunable intrinsic material properties, due to the sensitivity of the multi-scale structure of their constituent proteins to many environmental factors such as temperature, pH or ionic strength.17-20 Such unique properties imply that biological nanoobjects, especially the virus, can undergo subtle change at the molecular level under certain external stimuli, which might be amplified into some properties at the macroscopic scale.21 This is a big advantage of the biological nanoparticles comparted to the non-biological nanoparticles. Therefore, biological nanoobjects might be ideal building blocks for reconfigurable self-assembly.22-23 In the current contribution, we shall investigate the thermo-responsive in situ structure reconfiguration of a naturally assembled state - the chiral nematic liquid crystal (CLC) phase of some rodlike viruses, such as the M13 virus. The M13 virus particle consists of a helical assembly of many copies of small major coat proteins surrounding an elongated, single-stranded, circular DNA genome.24 The rodlike virus is a semi-flexible, cylindrical particle with a diameter of 6.6 nm and a length of ca.


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880 nm. Upon increasing concentration, the aqueous suspension of rodlike viruses will change from the isotropic liquid phase into the nematic liquid crystal (LC) phase, in which the long axis of the rodlike virus points to a general direction. The unique characteristic of M13 is that this virus actually forms a chiral nematic liquid crystal (CLC) phase (Figure 1).25-26 The rodlike viruses in such phase rotates helically along a given axis, resulting in well-defined optical “fingerprint” textures under polarized optical microscopy that relates to the cholesteric periodicity, i.e. pitch (P) (Figure 1). Although such three dimensional chiral arrangement of rodlike particles can be attributed to the intrinsic molecular chirality of the virus, some other rodlike viruses, such as the Pf1 virus with a chiral molecular structure highly similar to M13, could not form the CLC phase.27-28 Therefore, it seems that each kind of the viral building block encodes unique assembling information that dictates their specific self-assembling behavior in the nematic phase.7 Stimulated by such behavior, we conjecture that, for a given kind of viruses such as M13, their chiral ordering in the CLC phase might be tunable if there is some means to control the encoded unique assembling information of the viruses.29 Indeed, previous works by Fraden and Dogic have demonstrated that the degree of the chiral ordering of the M13 virus in the CLC phase can be influenced by temperature.27,

30

Such phenomena have further been

exploited by Dogic and coworkers in the polymorphic assembling of colloidal membranes consisting of rodlike viruses.9 However, there is no detailed investigation into such behavior and many questions are still open: Is the transformation from the chiral to less or non-chiral ordering truly reversible? Does there exit critical behavior?


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How will such parameters such as concentration (in the nematic phase range), ionic strength, exert influence on such behavior? Especially, the underlying mechanism of such chiral to non-chiral transformation is unclear. In the current work, systematic investigation will be presented to answer these open questions. Several analytical techniques, targeting levels ranging from the molecular, secondary conformation of the constituent proteins, to the whole single virus will be used to reveal subtle structure change of the coat protein that leads to such valuable 3D ordering transformation. These systematic investigations will be beneficial to fine-tune the thermo-responsiveness of such chiral to non-chiral ordering transformation which might have potential applications as one dimensional chiral materials or as matrix to regulate the ordering of other rodlike metal nanoparticles such as gold nanrods to create responsive metamaterials.31-34 In the last part of this work, preliminary results relative to the latter application will be presented.

Figure 1. Thermo-responsive changes of the fingerprint texture of the CLC phase of


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the M13 virus.

(A) Typical fingerprint texture of the CLC phase at low temperature.

(B) Texture of the nematic LC phase after incubating at 60 oC for several days. Schematic illustrations on the right are the arrangement of the rodlike virus in each phase. In (A), the pitch (P) is highlighted. The sample is 38.40 mg mL-1 M13 in PBS buffer (8mM, pH 8.0).

2. Experimental 2.1 Materials. Most of the chemicals were obtained from Sigma-Aldrich or J&K Company unless otherwise noted. 1-Anilino-8-naphthalene sulfonate (ANS) was obtained from Sigma-Aldrich and J&K Company respectively. All these reagents were used without any further purification. Water from a Millipore Milli-Q10 system (>18.2 MU) was used. The aqueous buffer mentioned in the work is a single batch of phosphate buffer (8 mM PBS, pH 8.0). The M13 virus was grown and purified following standard biochemical protocols using the E. coli ER2738 as the host bacteria.

2.2 Characterization of the cholesteric liquid crystal phase behavior by polarizing optical microscopy. The M13 virus samples were drawn into quartz capillaries with a diameter of 1.5 mm, both ends of which were then sealed with flame. The capillaries were cleaned with chromic acid and repeatedly rinsed with deionized water. Each sealed quartz capillary was filled with approximately 10 µL of the M13 virus suspended in PBS buffer (8mM, pH 8.0). The PBS buffer was used since pKa of such buffer has smaller temperature dependence. The samples in the capillaries were equilibrated for a few days at 4℃. The “fingerprint” textures characteristic of the CLC phase were observed on a Olympus-BX41 microscope equipped with


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LMPLanFL N lens (Olympus, LMPLanFL N) and recorded with a CCD camera (Micropublisher, 3.3RTV). The pitch was then analyzed with the software Image J. To realize heating and cooling, a home-made miniature planar water bath was used which can be used with the optical microscope. The temperature was controlled with an accuracy of 0.01 oC by an HAAKE A28 external circulating water bath installed with an HAAKE SC 100 controller (Thermo Scientific).

2.3 Ultraviolet Absorbance and Circular Dichroism Measurements. The concentration of the virus was determined by Ultraviolet (UV) absorbance spectroscopy on a UV-2550 UV-vis spectrophotometer (Shimadzu, Japan). The specific absorbance coefficient, including modest light scattering contributions to the apparent absorbance of the filamentous structures, at wavelength maxima was 3.84 cm2 mg-1 at 269 nm for M13. The circular dichroism (CD) spectra were collected on a MOS-500 Circular Dichroism Spectrophotometer (Biologic, France), equipped with Peltier temperature control units, to monitor the changes in ellipticity with temperature. Wavelength scans in the range of 200 - 400 nm were performed at different temperatures. The virus sample was suspended in PBS (8 mM, pH 8.0) at concentrations of 0.087 mg mL-1. Data were collected using quartz cuvettes with a 2 mm path length (Starna). Scans were performed at 50 nm min-1, 1.0 nm bandwidth, 4 s response, and 0.1 nm stepsize.

2.4 Fluorescence emission behavior of the M13 virus. The emission spectra of the intrinsic fluorescence due to the tryptophan residual of the pVIII coat protein and exogeneous fluorescence of ANS were recorded on a Hitachi F-4600 fluorescence


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spectrophotometer equipped with a thermostated cell holder. Samples were placed in a 10 mm × 2mm quartz cuvette. For the intrinsic tryptophan fluorescence, the excitation wavelength was set to 295 nm and the band widths for excitation and emission were set at 2.5 nm and 5.0 nm, respectively. The virus was suspended in 8 mM PBS (pH 8.0) at a concentration of 3.0 mg mL-1 and all the measurements were scanned from 300 to 400 nm at a scanning speed of 30 nm min-1, with three scans per sample. In the case of the ANS fluorescence, the excitation wavelength was 360 nm and the emission spectra were recorded between 390 and 700 nm; the excitation and emission slit was 10 nm and 20 nm, respectively.

2.5 Chiro-optical properties of gold nanorods regulated by the CLC phase of the M13 virus. Gold nanorods were synthesized using the classic seed-mediated growth method and were coated with poly(sodium styrenesulfonate) (PSS, MW = 70,000 g.mol-1) through layer-by-layer (LBL) deposition techniques to make the surface of the nanorod negatively charged.35 The zeta potential of the initial gold nanorods was about +41.18±0.76 mV due to the bilayer of CTAB molecules on the surface. The PSS-coated gold nanorods showed a zeta potential of − 30.68 ± 1.81 mV. The extinction spectra of GNRs with or without PSS and GNRs dispersed in the M13 virus were recorded in the range of 400 to 900 nm. To 500 µL M13 suspension (47.81 mg mL-1, in 8 mM PBS buffer) was added 50 µL GNR suspension and the resulted mixture was thoroughly mixed. The concentration of GNR in the final mixture is 1.04

× 10-9 mol L-1. Part of the sample was sealed into a quartz capillary with a diameter of 1.5 mm and observed with POM to check the formation of the CLC phase. Another


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part of sample was placed into a rectangular sample cell made of quartz specific for the CD measurement and with a thickness of only 100 µm. The extinction spectra of the sample were recorded in the range of 400 to 900 nm. The CD measurements of the sample, with the polarized light normal to the surface of the rectangular sample cell, were carried out in the range of 400 - 950 nm.

2.6 Characterization. The gold nanorods were checked by transmission electron microscopy (TEM) measurements on a Philips T20ST electron microscope at an acceleration voltage of 200 kV. To prepare the TEM samples, the gold nanorods solution was dropped onto a carbon-coated copper grid and dried slowly in air. Zeta potential measurements of the particles were conducted on a Malvern Zeta Sizer (Nano ZS) at room temperature. The particle size of the virus was monitored on a laser light scattering goniometer (BI-200SM) equipped with a digital correlator (BI-10000AT) and a laser source of 532 nm. All of the samples were filtered through a 0.45 mm Millipore filter into clean scintillation vials right before characterization. Data were collected at scattering angles of 18°. Conditions were held at different temperatures, ranging from 20℃ to 60℃, and a concentration of 7.5 × 10-5 mg mL-1, well below the overlap concentration.

3. RESULTS AND DISCUSSION 3.1 Thermo-responsiveness of the Cholesteric Liquid Crystal (CLC) Phase of the Rodlike M13 Virus. Typical cholesteric fingerprint textures can be observed by polarizing optical microscopy (POM) in the concentrated suspensions of the M13 virus after equilibrium for a few days at room temperature (Figure 1A). In the


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cholesteric textures, the distances between the adjacent dark and bright stripes in the fingerprint patterns are equal to 1/2 the cholesteric pitch, P, which represents the twist periodicity and twisting power.27, 36 P was monitored upon heating and cooling for several samples in the temperature range of 10 to 60 oC. The sample was equilibrated at each temperature at least for two hours until the measured values of P approach constant, indicating the system approaches equilibrium. As listed in the Figure 2, upon heating up to 40 oC, P increased moderately with increasing temperature. After this temperature, a sharp increase of P occurred. Incubating at 60 oC for some time, the fingerprints in the capillary eventually disappears, suggesting the system turns into the normal nematic LC phase, i.e. the long axis of all viruses point to one direction but without positional ordering. The turning points of P versus temperature is always occur at ca. 40 oC, independent of the ionic strengths and virus concentrations. This is surprising, since low ionic strength and high virus concentration mean higher viscosity,37 which might hinder the re-ordering the rod from the chiral arrangement into non-chiral ordering. This probably points to the fact that some intrinsic properties occur to the virus itself around this temperature. It is noted here that the concentration of the virus was far-away from the nematic-sematic phase transition boundary.38 The increase of pitch cannot be due to pre-sematic unwinding.27 From light scattering performed at the small scattering angel, the narrow distribution of the apparent hydrodynamic diameter in is almost constant upon heating (Figure S3 in SI), indicating no aggregation occurs to the virus during the temperature range of interest. Therefore, rearrangement of the rodlike particles relative to each other in three


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dimensions was purely induced by heating stress.

Figure 2. Dependence of the cholesteric pitch on temperature upon heating and cooling. The arrow highlights the critical temperature at which the increasing or decreasing rate changes. (A) 35.50 mg mL-1 M13 in PBS buffer (8 mM, pH 8.0, 100 mM NaCl was added.). (B) 38.40 mg mL-1 M13 in PBS buffer (8 mM, pH 8.0). (C) 62.45 mg mL-1 M13 in PBS buffer (8 mM, pH 8.0). (D) Pitch versus time during fast heating (10 60 oC) and quenching (60 10 oC) experiments. The sample is the same as in (B) and was swiftly changed from one temperature to another. Inset: the pitch change upon cycling of heating/cooling. Up to 60 rounds of heating/cooling were performed and only part of the results is shown here.

The chiral to non-chiral ordering transformation is highly reversible in the temperature range investigated here. By cooling back, P decreases to the value before heating and P versus temperature identically follows the trace of heating, with a turning point at 40 oC. The heating/cooling cycles can be performed for many rounds


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(Up to 60 cycles were performed and part of the data is listed in the inset of Figure 2D, also Figure S1 in SI).

We have to point out that the temperature investigated herein

is in the range of 10 ~ 60 oC. As discussed later, we found by circular dichroism (CD) that, irreversible change occurs to the secondary structure of the coat protein at 75 oC and concentrated virus suspension incubated at this temperature or above cannot revert into the CLC phase. In the temperature range up to 60 oC, the virus can keep structure integrity and are still infection active.39-40 The time scale of the chiral to non-chiral ordering transformation or vice versa was investigated by fast heating and quenching experiments, respectively. A capillary containing a concentrated virus suspension which formed a perfect CLC phase was quickly submerged into a water bath pre-heated at 60 oC. On the contrary, in the quenching experiment, a capillary containing the same concentrated virus suspension without any chiral ordering by incubating at 60 oC for several days is submerged into a water bath at 10 oC. The change of the fingerprint texture during these procedures was recorded with a CCD camera, resulting in movies which unambiguously reveal the amazing LC texture variation during quick heating/cooling (Movies in the Supporting Information). The pitch versus time during these procedures was determined and listed in Figure 2D. During fast heating, P sharply increases to a constant value during ca. 300 seconds. Further incubating for ca. five mins, the cholesteric texture eventually disappeared and there was only homogenous birefringence due to the normal nematic LC phase. During quenching, fingerprints quickly appeared in the first 5 seconds, the time scale needed for preparation of the camera recording. P versus time during quenching exhibits distinct behavior compared to the quick heating. The pitch sharply decreases during the first 100 seconds, goes through a gradual decrease and then finally approaches the value of the sample at room temperature during 700 seconds (Figure 2D). In summary to this section, detailed investigations reveal many precious


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characteristics of the thermos-responsiveness of the current system. The 3D chiral ordering can transform into a non-chiral ordering induced by heat stress. The procedure is highly reversible in certain temperature range and can be repeated for many times. There exist a critical temperature around 40 oC which is independent of the ionic strength and virus concentration. The time scale is reasonable. These properties are extremely important material properties for intelligent materials in terms of reusability, sustainability and low maintenances.12, 41

Insight into the reversible chiral to non-chiral ordering transformation by techniques targeting different structure level. As mentioned before, one of the puzzles surrounding the rodlike virus is that viruses with highly similar molecular and subunitary structure will form distinctly different nematic phases.28 For instance, while M13 forms the chiral nematic phase, other viruses such as Pf1 can only form pure nematic phase. Previous investigations from several groups have come to the conclusion that such behaver might be due to subtle difference in the following aspects such as interaction of the coat protein and DNA/RNA, surface properties such as charge patterns, hydrophilicity/hydrophobicity, etc.28, 36, 42-43

In the above section,

it is demonstrated that different nematic phases can be realized with the same virus under heating stress which probably induces the changes of the factors that contribute to the different nematic phases formed by viruses with similar molecular structures. Therefore, from now no, several analytical techniques will be used to shed some light on the connection of the structure change of the viruses at various levels during heating or cooling to the chiral to non-chiral ordering transformation.


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Scheme 1. Molecular structure of the M13 virus and illustration of three analytical techniques which can sense different levels of structure change. The α-helical pVIII (B) and part of the M13 Virus (C) is drawn using PyMOL based on the updated model (PDB ID code 2MJZ). In (A) is the amino acid sequence of pVIII.

The main structure feature of the M13 virus is a protein capsid consisting of 2700 identical copies of the major coat protein, pVIII, which arranges in a helical way in the capsid (Scheme 1).24 At each end of the virus is terminated by several copies of minor proteins (pIII, pVI, pVII, pIX). A single-stranded circular DNA sits inside the capsid. pVIII is a short peptide containing 50 amino acids and has a largely α-helical conformation. Starting from the N-terminal, this protein can be roughly divided into three segments with different functionalities: the first 1-20 amino acid residuals are exposed to the solvent, have an amphipathic nature and dictate the surface property the virus; the middle segment, containing the 20-40 amino acid residuals, is hydrophobic and also the location for the inter-protein packing into the cylindrical capsid via hydrophobic interactions; the rest of 40-56 amino acid residuals at the


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C-terminal is the third segments which is positively charged and interacts with the encapsulated DNA. It is well-established that the hydrophobic middle segment is the main determinant of the inter-subunit packing and responsive for the stability of the whole virus.44 With such structure feature in mind, the following three analytical techniques were exploited to monitor the subtle structure change upon heating and cooling: 1) Intrinsic fluorescence of the tryptophan residual of pVIII at the positon of 26 (W26) which can detect the local environment change at the single molecular level. 2) Circular dichroism spectroscopy (CD) that can reveal the change of the α-helical conformation of pVIII. 3) ANS fluorescence that sense the surface properties the virus at the single virus level. Intrinsic fluoresce of the tryptophan residual of the coat protein. The single tryptophan residual (W26) in the middle hydrophobic portion of pVIII plays a paramount key role in the packing of PVIII into the capsid.44 Found in the coat protein of most of filamentous viruses, this residual is highly conserved. Coat proteins with W26 replaced with other amino acids by genetic mutagenesis cannot self-assemble into the helical capsid. Recent work down to the atomic scale indicates that W26 has direct contacts, through either hydrogen bonding or π-π stacking, with several other amino acid residuals from adjacent protein subunits.45 Therefore, W26 can be used as a diagnostic handle to sense the delicate structure change of the virus under external stresses at the single amino acid level. Fortunately, under an appropriate excitation wavelength, the M13 virus has significant intrinsic fluoresce due to W26, which is extremely sensitive to its local surrounding environment.46 To investigate the intrinsic tryptophan fluorescent emission, the M13 suspension was exited at 295 nm to avoid any interference from other aromatic residuals such as tyrosine since the extinction coefficient of tryptophan at 295 nm is considerably larger than that of tyrosine. As listed in Figure 3, there is strong fluorescent emission with a maximum peak locating


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at 328 nm at 10℃, in line with the fact that W26 is sequestered in the hydrophobic domain of the inter-subunit (Scheme 1). Upon heating from 10 to 60 ℃ , the fluorescent intensity sharply decreases, accompanied by a slight blue-shift of the maximal emission peak from 328 nm at 10 oC to 326 nm at 60

o

C (Figure 3C). Such

blue-shift, although to a very slight degree, usually implies the local environment of tryptophan becomes even more apolar.47 It is noted that the blue shift occurred at ca. T = 40 oC, consistent with the turning point of the pitch versus temperature.

Figure 3. Intrinsic tryptophan fluorescent emission behavior of the coat protein-pVIII of M13 during heating and cooling. (A) and (B) fluorescent emission spectra upon heating (A) or cooling (B) in the temperature range of 10 – 60 oC. The spectra were recorded after excitation at 295 nm. The sample was 3.0 mg mL-1 M13 suspended in PBS (8mM, pH 8.0). (C) and (D) Maximum emission peak (C) and intensity at each peak (D) versus temperature upon heating and cooling.

Of special notion is the pronounced decrease in the quantum efficiency of W26 upon heating, as manifested by more than two order of magnitudes of decease in the fluoresce intensity upon heating from 10 to 60℃(Figure 3D). Such phenomena are


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probably due to quenching by several mechanisms,48 among which, the excited-state proton transfer to other aromatic residuals, such as tyrosine or phenylalanine at position 21 or 45 from adjacent protein subunits, might be highly possible.45,

49

Heating-induced tiny structure change of pVIII and subsequent protein-protein packing alteration in the capsid can facilitate the direct contact of these aromatic residuals, resulting in quenching of the tryptophan fluorescence emission.49 The change of the tryptophan fluorescence emission is highly reversible, cooling back to the initial temperature restores both the intensity and the emission maximum peak (Figure 3B). Such behavior is consistent with the reversible transformation between the chiral to non-chiral ordering state. Circular dichroism of the coat protein. Intensive investigations in the past decades have confirmed that the major coat protein, pVIII, of M13 and similar viruses have a largely α-helical conformation.50-51 Herein, the secondary structure of pVIII was monitored by circular dichroism (CD) spectroscopy upon heating and cooling (Figure 4). The signal from the CD spectroscopy is dominated by that due to pVIII since this major coat protein accounts for 87% of the entire viral particle on a mass basis.51 The unique property of the CD spectrum of M13 and similar viruses at room temperature is that there is a shoulder at 208 nm and a peak at 222 nm, which stems from the α-helical conformation of pVIII and intermolecular π-stacking between the tryptophan and phenylalanine residual at position 26 and 45 respectively.46 Increasing temperature causes a concomitant decrease in the intensity of the characteristic 208 nm and 222 nm (Figure 4A). According to previous work, decrease of ellipticity at


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208/222 nm of pVIII as the temperature increases results from the transition from α-helix to β-sheet.17 In other words, the helical structure of pVIII unwinds slightly during heating. The compositional fraction of α-helix of pVIII is estimated following literature, which reduces from 96.3% at room temperature to 81.9% at 60 oC (Figure 4C).51-52 Since the origins of circular dichroism can be traced to the direct intermolecular stacking of tryptophan (W26) upon phenylalanine (F45) residues of pVIII,46 unwinding of the coat protein is expected to influence such stacking and also the local environment of W26. This is consistent with the heat induced change of the intrinsic fluorescence of tryptophan residual (W26). By cooling, the CD spectra recover to the original state and does also the compositional fraction of the α-helix, indicating the secondary structure change of pVIII induced by heating is reversible in the temperature range of the current work. As mentioned before, upon monitoring the ellipticity at 222 nm during heating, there was a dramatic increase around 70 oC and the ellipticity could not be recovered to the value at lower temperature (Figure 4D), indicating irreversible structure change occurred after this temperature.53 There also exists a clear turning point locating at 40 oC.


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Figure 4. Circular dichroism (CD) spectra of the M13 virus upon heating (A) and (B) cooling. MRE refers to mean residual ellipticity. The virus concentration is 0.087 mg mL-1 in PBS buffer (8mM, pH 8.0). (C) α-helical conformational fraction of the major coat protein-pVIII versus temperature. (D) Ellipticity at 222 nm versus temperature upon continuous heating. The speed of increasing temperature is 0.5 oC per min.

ANS fluorescence at the surface of M13 virus at the single virus level. As stated before, each M13 virus consists of 2700 identical copies of pVIII which self-assembles and packs into a cylindrical capsid in a helical way, mainly through hydrophobic interactions of the middle hydrophobic segment of pVIII (Scheme 1). It is reckoned that the secondary structure change of pVIII upon heating, as confirmed by CD, might also influence the inter-protein packing in the capsid which can be revealed by the 1-anilino-8-naphthalene sulfonate (ANS) fluorescence. ANS is an amphiphilic fluorescence dye which is non-fluorescent in aqueous solutions but becomes intensively fluorescent when binding to hydrophobic interfaces or in


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non-aqueous environments.54 Classical studies have shown that viscosity and polarity of the local environment surrounding ANS determine the characteristic emission maximum and intensity of ANS fluorescence. Such valuable properties make ANS a popular dye to detect the surface hydrophobicity of proteins or cell membranes, and conformation change of a protein during denaturation. The ANS fluorescence spectra in the presence or absence of the M13 virus and upon heating/cooling are summarized in Figure 5. In the case of ANS alone in aqueous buffer, the ANS fluorescence is very weak with an emission maximum (λmax) locating at 512 nm (Figure 5A). In contrast, the emission intensity of ANS in the presence of M13 increases dramatically and λmax blue shifts from 512 to 473 nm, suggesting strong interaction of ANS with the virus. Such interaction probably has the electrostatic nature since the amino group of lysine at the position of 8 of pVIII might interact with the sulphate group of ANS. Only electrostatic interaction, however, could not induce the strong blue-shift and enhanced quantum efficiency of ANS. Therefore, it is highly possible that ANS is binding with the hydrophobic surface by penetrating into the membrane like hydrophobic domains of inter-protein subunits in the protein capsid of M13.55 Upon heating from 20 to 60℃, the ANS fluorescence intensity decreases with a degree of three order of magnitudes, along with a red shift of the emission maximum from 469 to 477 nm (Figure 5A). Decreasing fluorescence intensity together with red-shift usually means the local environment of a choromphore, here, ANS, become more hydrophilic. Therefore, the environment around ANS became relatively more hydrophilic at elevated temperatures compared with that at lower temperatures. Since ANS mainly resides in


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the inner hydrophobic domains of inter-protein subunits of the capsid, change of its surrounding environment can only be brought by the variation of the packing style of adjacent pVIII coat proteins. It is noted here that the fluorescent emission behavior of ANS is again reversible in the temperature range of 10 to 60 oC. Cooling back to room temperature restored both the emission maximum and also the intensity (Figure 5B).

Figure 5. ANS fluorescence behavior in the presence of the M13 virus upon heating and cooling. The virus concentration is 3 mg mL-1 in PBS buffer (8mM, pH 8.0) contain 0.02 mg mL-1 ANS. (A) and (B) Fluorescent spectra of ANS in the presence of M13 virus during heating (A) and cooling (B). Spectra of pure ANS and M13 at room temperature are also listed in (A). (C) and (D) Maximum emission peak (C) and intensity at each peak (D) versus temperature upon heating and cooling.

Mechanistic insight into the heating induced reversible chiral to non-chiral ordering transformation in the nematic phase of M13. For the convenience of the


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following discussion, we first note here that the two viruses, fd and M13, are similar to each other with only one amino acid residual difference at the position of 12 of the solvent exposed part of the coat protein.24, 44, 50 In addition, the two viruses have identical structure and similar liquid crystal behavior under the same conditions. Therefore, we should not distinguish them from each other in the following discussion. Pf1 is another filamentous virus that has also been subjected to intensive investigations.19 The amino sequence of the coat protein of Pf1 are different from the fd or M13 virus, so does the packing style of the coat protein.24 The above filamentous viruses, together with many other filamentous viruses, are intrinsically chiral: they are helical assemblies consisting of several thousands of identical major coat protein subunits with an α-helical conformation arranging in a helical way (Scheme 1). However, some viruses such as M13 and fd, form a chiral nematic LC phase while others, like Pf1 can only form a pure nematic LC phase.28 Several speculations have been proposed to explain such intriguing phenomena by Fraden, Zvonimir and Grelet, among which the superhelical assumption has been supported by many evidences.36, 43, 56 Rather than the rodlike shape with a straight contour normally appearing under electronic microscopy or atomic force microscopy, some kinds of viruses might exist in the aqueous suspension state as a superhelical twist, or a dynamical “cork-screw” shape. It is this large-length-scale chirality that is responsible for the formation of the cholesteric nematic LC phase since two screws can achieve maximum contact by approaching each other at certain angle. Day and Green further argue that whether or not one virus can coil into such a superhelical


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shape is determined by the interaction between the viral DNA and the coat protein subunit.28 Incommensurate interaction between the two components produces certain internal stress that forces the virus to coil, resulting in subunit polymorphism. In another word, the chemically identical pVIII proteins are indeed in different environments. NMR studies by Opella have confirmed the conformational heterogeneity of pVIII in the capsid of fd or M13.49 This is consistent with the fact that subunits on the inside of any coil are closer to one another than those on the outside. However, similar subunit polymorphism has not found with the Pf1 virus, which has a 1:1 commensurate DNA and coat protein interaction. Therefore, fd or M13 are probably in the coiled state in suspension while Pf1 adopts a strait conformation. Recently, Dogic and coworker have investigated the LC behavior of a mutant of the fd virus, fd Y21M, of which the tyrosine amino acid residual at position 21 is replaced by methionine. fd Y21M forms a chiral LC phase with a pitch much larger than the fd virus under identical conditions.43 Such behavior is coincident with the fact that the former has a much larger persistence length than the later. This means that the former is much stiffer than the later. The authors suggested such behavior might stem from the packing order difference of pVIII in the cylindrical capsid. By replacing tyrosine at position 21 with methionine which is exactly in the location responsive for the inter-subunit packing, some inter-subunit hydrogen bonding is interrupted in the case of fd Y21M.49 The pVIII subunit can undergo repacking into a more regular ordering, leading to a less coiled conformation. Besides this work, Day and Green also showed


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that the surfactant SDS can induce some viruses into a CLC phase from a normal nematic one.28 It is believed that SDS can penetrate into the inter-coat protein of the capsid and influence the packing of pVIII. These elegant results together point to the fact the inter-protein subunit packing can be tuned on purpose by external stimuli without disturbing the DNA-coat protein interactions. The current work further demonstrates that heat stress can be explored to tune the coat protein ordering which can further amplify into a macroscopic reordering of the virus particles in the nematic phase. And the whole procedure is highly reversible in the temperature range investigated here. With the above three techniques that target information at different levels, it is clear that heating induces slight conformation change of the coat protein subunit, and therefore the packing of the coat protein inside the capsid. First of all, the local environment of W26 in the middle hydrophobic section of pVIII is changed by heating. W26 is responsible for the hydrogen bonding with several amino acid residuals such as T21 from adjacent coat proteins. Such bonding is believed to play a critical role in the formation and stabilization of the protein capsid. Furthermore, the CD indicates the α-helical conformational fraction of pVIII is 93% at room temperature and decreases to the 80% at 60 oC, suggesting unwinding of the coat protein. Coincidently, the α-helical conformational fraction of pVIII of the Pf1 virus is also around 80% at room temperature at which this virus cannot form the CLC phase.51 Such unwinding might change the relative position of the amino acid residuals, resulting in the breakup of the hydrogen bonding of W26 with neighboring


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amino acid residuals. Rearrangement of the coat proteins relative to each other on a single virus level is possible, as revealed by the fluorescent emission behavior of ANS which originally locates in the hydrophobic area of inter-coat protein of the capsid. The three complementary techniques reveal that repacking of the coat proteins clearly occurs, to a very subtle degree without disrupting the virus integrity. The deviation of the coat protein from the α-helical conformation and the interruption of hydrogen bonding facilitate the repacking of pVIII into a more regular ordering, just like in the case of the Pf1 virus or fd Y21M.19 Such changes in the packing state force the virus to become more rigid and the virus change from the more coiled superhelical state into a less coiled state. The conformation change of the coat protein and their rearrangement relative to each other in the helical capsid, without disrupting the structure integrity of the overall virus, can be generally classified as polymorphism.18-20 Polymorphism seems a common trait of many biological particles, especially viruses. For instance, many icosahedral viruses undergo structure change at the single virus level, such as compacting and spatial reordering of their coating protein in the maturation into the matured virus. In addition, it was reported that Pf1 and Pf3 viruses can also undergo polymorphism during cooling, concentration or even changing of ionic strength. Such polymorphism normally involves concert movement of several thousands of individual proteins and seems vital to the survival of the many filamentous viruses, which might when facing many unforeseen situations such as shearing, heat stressing, etc. Indeed, the coat protein of the bacteriophages which replicate through infecting


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the bacterial host, has to go through several different stages in the life cycle of a virus.57 The coat protein-PVIII first stays in the cell membrane of the host in a conformation higher in β-structure. In the next step, the coat protein moves out the membrane and self-assembles onto DNA to form the final mature rodlike virus in which the coat protein is in the dominant α-structure. In the infection, the procedure is reversed. Therefore, the PVIII is very flexible in the structure change for the sake of the propagation of the virus. Therefore the survival stagey of a biological organism can be transformed into very precious material properties.

Handedness of the CLC phase of M13 by induced circular dichroism of doped gold nanorods. Handedness is another critical parameter of the CLC phase. Grelet has developed a method to characterize the handedness of the CLC phase of rodlike viruses via monitoring the configuration of a few fluorescently labeled viruses in a background of well-developed CLC phase of unlabeled viruses.56 In the field of molecular LC, circular dichroism (CD) is a method of choice for sensitively revealing the handedness in a non-invasive way.58 CD measures the difference in the absorption of left- and right-handed circularly polarized light as a function of wavelength as the light passes through an optical active medium, such as the CLC phase. However, the chiral nematic pitch of the M13 suspension is in the range of more than decades of µm and is much longer than the wavelength of visible light. Therefore it is impossible to determine the handedness directly from the sign of circularly polarized light reflected by the sample. Alternatively, a CD signal can be induced by the association between a CD inactive chromophore and the chiral LC structure.59 If the chromophore


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has an anisotropic shape, their arrangement in the host can assume the same handedness of the host CLC phase. Therefore, the induced circular dichroism (ICD) can reflect the handedness of the host.60 Herein, we used gold nanorods (GNRs) as the dopant to derivate the ICD of the CLC phase of M13.34 Although the results presented here are highly preliminary, we aim to 1) develop an alternative way to determine the handedness of the CLC phase of the virus and 2) demonstrate the potential of the virus based thermo-responsive CLC phase as the matrix to regulate the chiro-optical properties of the 1D noble metal nanoparticles (Figure 6A).3, 34

Figure 6. CLS phase of the M13 virus doped with gold nanorods (GNRs). (A) Schematic illustration of the possible arrangement of GNRs in the chiral environment of the CLC phase of the virus. Long purple rods are viruses while short yellow rods are GNRs. (B) Transmission electron microscopy of GNRs. (C) Typical fingerprint texture of the CLC phase of the M13 virus doped with GNRs. (D) Pitch versus temperature upon heating and cooling. The sample consists of the M13 suspension


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(47.81 mg mL-1, in 8 mM PBS buffer) doped with 1.04 × 10-9 mol L-1 GNRs.

Gold nanorods exhibit surface plasmon resonances (SPRs) and possess two distinct plasmon bands associated with the asymmetric structure of the materials: one, known as the transverse localized SPRs (TLSPR), corresponds to the transverse axis, and another corresponding to the longitudinal axis, termed the longitudinal localized SPRs (LLSPR).61 Figure 6B is the typical TEM images GNRs used here with an average length of 45 nm and a diameter of 12.5 nm. The surface of GNRs was negatively charged by coating them with poly(sodium styrenesulfonate) in order to facilitate mixing of the M13 virus with GNRs. There only exists repulsion between the two kinds of rods. The extinction spectra of the mixture of GNR and M13 exhibit characteristic TLSPR and LLSPR at 528 and 705 nm, respectively (Figure S2 in SI), which are similar to the GNRs alone except slight red-shift, indicating no aggregation of GNRs in the mixture.34 Upon heating, the peaks of LLSPR of the GNRs moved to longer wavelengths (Figure 7A), accompanied with no obvious shift of TLSPR, as a consequence of the local environment changes or concomitant effect of the plasmon coupling between GNRs.34


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Figure 7. Extinction spectra of GNRs (A) and induced circular dichroism (ICD) GNRs in the CLC phase of the M13 virus (B) upon heating. The sample consists of the M13 suspension (47.81 mg mL-1, in 8 mM PBS buffer) doped with 1.04 × 10-9 mol L-1 GNRs.

The concentrated M13 suspension, doped with GNRs could still form the CLC phase and the pitch change upon heating/cooling is similar to those without GNRs (Figure 6C and D). The GNRs doped M13 suspension was placed into a rectangular sample cell made of quartz specific for the CD measurement and with a thickness of only 100 µm. The boundary conditions imposed by the cell surfaces ensure that twisting occurs


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along a helical axis perpendicular to the cell surface (Figure 6A). The GNR suspension alone has no clear CD signal in the range of 400 to 850 nm (Figure 7B). The M13 suspension without GNR shows some very weak CD signals with a negative value and large noises. In contrast, The GNR doped M13 virus suspension in the CLS phase exhibits two positive CD peaks, typical of a left-handed chiral nature (Figure 7B).34 The wavelength of the two peaks are in the correspondence with the two extinction peaks of the GNRs, indicating that the CD peaks are due to the presence of GNRs in a chiral environment. Most importantly, although the CD peak at position 705 nm is most identical to the extinction peak due to LLSPR (Figure 7B), the one at position 450 nm is blue-shift from the extinction peak at 528 nm due to TLSPR. Such blue-shift is attributed to the coupling effects between two neighboring GNRs with chiral arrangement, termed chiro-optical effect. Therefore, these results together confirm that the orientation of the major axis of GRN mirrors that of CLC phase of the M13 virus (Figure 6A). Upon heating, the intensity of CD signal at both peaks decreases, due to the decreasing long-range chiral periodic order of GNRs which diminishes the induced plasmonic chiro-optical activity of the NRs. Further heating induced the separation of the GNRs from the virus suspension, which makes further investigation impossible. Future work will focus on tackling such issue in order to create truly tunable chiro-optical metamaterials by exploiting the thermos-responsive CLC phase of rodlike viruses.

4. CONCLUSION Detailed investigations presented herein confirm that the heat induced transformation


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from the chiral to less or non-chiral ordering in the CLC phase of M13 virus is highly reversible in certain temperature range. Around temperature 40 oC, the increasing or decreasing rate in the pitch versus temperature changes dramatically. This critical temperature is independent of ionic strength and virus concentration (in the nematic phase range). Fast heating and cooling experiments reveals the time scale of such transformation is in the range of several mins. Subtle secondary conformational structure change of the coat protein – pVIII and their packing style in the virus capsid have been revealed by several techniques, targeting levels ranging from the single tryptophan amino acid residual, secondary conformation of the constituent proteins to the whole single virus, respectively. Such subtle change might be relative to the superhelical state of M13 in the suspension sate. Induced circular dichroism of gold NRs doped inside the CLC phase of M13 reveal the left-handedness of the phase. The chiro-optical properties of the gold NRs can also be regulated by the heat-induced chiral to non-chiral ordering transformation.

ASSOCIATED CONTENT

Supporting Information

Extinction spectra of GNRs under different conditions, movies of the fingerprint change upon quick heating and cooling, this material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors


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E-mail: [email protected]. Fax: +86 22 23503510. Tel: +86 22 23501945

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21274067, 91127045, 51390483), the Fundamental Research Funds for the Central Universities, Natural Science Foundation of Tianjin, China (No. 12JCQNJC01800) and PCSIRT (IRT1257). ZZK thanks Prof. Linqi Shi for his generous supporting.

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Thermoresponsive Chiral to Nonchiral Ordering Transformation in the

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