Biophysical and Amyloid Behaviour of a Recombinant Rab1 Isoform of Leishmania Donovani

doi:10.21203/rs.3.rs-2834986/v1

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Biophysical and Amyloid Behaviour of a Recombinant Rab1 Isoform of Leishmania Donovani

https://doi.org/10.21203/rs.3.rs-2834986/v1

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The most lethal form of leishmaniasis in mammals ^_ kala-azar or visceral leishmaniasis, is caused by the intracellular protozoan parasite Leishmania donovani. The several phases of infection and life cycle of the parasite are regulated by Rab proteins, a small GTPase subfamily. This study uses biophysical methods to describe the pH-moderated solution characteristics of a recombinant Rab1 isoform from L. donovani (rLdRab1). The isoform unfolds on lowering the solution pH in a non-two-state route, highlighting the potential for more than one folding intermediates. At pH 2.0, a partially folded intermediate (PFI) state accumulates and exhibits properties median to the molten and pre-molten globule states. At high protein concentrations the high amyloidogenic potential of this PFI induces slow transformation into distinct protofibrils. It was also noted that the native state of the isoform was aggregation-prone. Nevertheless, amorphous aggregation rather than fibrils was formed. This is the first work, to our knowledge, to describe the amyloid-like behaviour of Rab isoforms in L donovani in vitro. This research provides an unusual alternative to comprehend the full biophysical properties of the Rab1 isoforms in the lower eukaryote L. donovani.

Visceral leishmaniasis

Recombinant Rab1

protofibrils

protein aggregation

Circular Dichroism

A group of intracellular parasites of the Leishmania genus are the etiological agent of several leishmaniasis disorders. Visceral leishmaniasis (VL), its most severe form, is primarily endemic to countries in South Asian and African regions. [1]. The infection is propagated through the vector sand fly. Following the injection of the flagellated promastigote type of the parasite in human circulation, it replicates into the immobile amastigote form inside the host macrophages. The infection is subsequently disseminated by the amastigotes invading and proliferating inside fresh host cells. [2, 3]. Rab proteins in Leishmania regulate and mediate the exocytic and endocytic processes required for the infective propagation [3, 4].

A vast majority of small GTPase Rab subfamily members belong to the Ras G protein class [5, 6]. Rab proteins are ubiquitous in eukaryotes governing all levels of vesicular traffic. There are ~ 60-70 Rab proteins identified in humans, 11 in yeast, 16 in lower protozoans including Leishmania [6–9]. The GTPase fold is a characteristic evolutionary conserved domain identified in all Rab proteins. This fold has a six-stranded sheet and five helices that are joined by 10 loops. The protein is directed to its destination by the hypervariable region at its C-terminus [5–7]. In the cytoplasm, Rab is coupled to the GDP molecule in an inactive state. Following a series of events, Rab is transformed into an active GTP-bound state that is recognised by downstream effector molecules. [5–7].

Molecular functions of four Rab proteins of L. donovani are experimentally described. [10–12]. Biophysical chemistry aspects of only a few protozoan Rabs have been explored [10]. Solution behaviour and conformations determine the functional characteristics of proteins. Hence, it becomes intriguing to study the biophysical chemistry of proteins. In this work, we have cloned and purified a putative Rab1 from L. donovani (rLdRab1) and characterised its structural features using in silico tools and biophysical methods. Secondary and tertiary structures were investigated using Far-UV circular dichroism and fluorescence spectroscopy. The pH-induced unfolding study indicated accumulation of an intermediate state (I) around pH 2.0. The intermediate was found to be highly aggregation prone and formed stable amyloid-like protofibril. Rab GTPases shuttle between the cytosol and membranes and are thought to be highly susceptible to aggregation in the cytosol [13, 14]. Hence, this study provides a novel opportunity to understand the unfolding and aggregation behaviour of the Rab protein in Leishmania.

2.1. Materials

Guanidine hydrochloride (GuHCl, G3272), 1-Anilino 8-naphthalene sulphonic acid (ANS, A1028), Thioflavin T (ThT, T3516) and Congo Red (CR) were purchased from Sigma-Aldrich Co. USA. All other reagents used were of analytical grade with purity >99%. All the media, buffers and solutions were prepared in autoclaved sterile milli-Q water in pre-sterilized glassware. Biophysical chemistry experiments were performed in a 60 mM sodium phosphate buffer for pH range 6-8, 75 mM glycine -HCl buffer for pH range 1.5-3.0- and 60 mM acetate buffer for pH range 3.5-5.5. The pH was checked using Cole Parmer P200 benchtop pH meter (Cole Parmer, United States).

2.2. Production of recombinant protein

The protein sequence of LdRab1 was retrieved from L. donovani database [15, 16], PCR amplified and cloned into pGEM-T vector as described earlier for other proteins [17, 18]. The production and purification of recombinant protein is described in supplementary information.

2.3. In-silico studies

The sequence of the cloned recombinant protein was given as the input query for in silico prediction studies. Domain analysis was carried out using sequence alignment by BLAST search engine (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Physical characteristics were predicted by ProtParam tools using Expasy server [19]. Three-dimensional model of the protein was developed using the Swiss Model server [20]. The secondary structure predictions were carried out using PHYRE [21], predict protein [22] and JPRED [23] .

2.4. pH induced unfolding

Purified rLdRab1 was diluted to 2 μM in buffer solutions of pH range 1.5 to 8.0, at intervals of 0.5. The samples were allowed to equilibrate at room temperature for ~4 hours. Intrinsic fluorescence (IF) and ANS fluorescence were recorded as described in section 2.6 and 2.7 respectively. Fluorescence data points are represented as mean intensities of IF at 340 nm and ANS fluorescence at 480 nm as a function of pH. The resulting unfolding curves were fitted using non-linear regression Eq. 1[24].

Where, f_U, f_N represent fluorescence signals of unfolded and native state at pH (x), x₀ represents midpoint of the transition and b is the slope factor of transition.

2.5. Circular dichroism (CD)

2 μM purified rLdRab1 prepared in pH 6.5, pH 2 and 6 M GuHCl was incubated for 4 hours at room temperature for denaturation. The reaction mixtures containing final aggregation products (section 2.8) were diluted to 2 μM in phosphate buffer at pH 6.5. Far UV-CD of the protein was recorded between 200 and 250 nm on Jasco 815 Spectropolarimeter with Path length and slits fixed at 3 mm and 1 nm, respectively. The data were presented as mean residue ellipticity ([θ], MRE) expressed in deg cm² dmol^-1. Eq 2

where CD is in millidegree, n is the number of amino acid residues, l is the path length of the cell in cm, and C_p is the molar concentration of the protein.

2.6. Intrinsic fluorescence (IF)

The rLdRab1 solutions prepared in section 2.4 and 2.5 were excited 280 nm. The IF spectrum was collected between 300 and 450 nm on the Spectramax M2E spectrofluorometer (Molecular Devices). To collect Tryptophan fluorescence, the samples were excited at 295 nm and emission was recorded between 310 to 450 nm. The path-length was fixed to 1 cm with fixed excitation and emission slits of 5 nm each.

2.7. ANS assay

ANS assay was performed as described earlier [25]. Briefly, to 2 μM rLdRab1 (section 2.4, 2.5) ANS was mixed to obtain a 20 μM ANS-protein solution. For tinctorial characterization of aggregates (section 2.8), the reaction mixtures containing final aggregation products were diluted to 2 μM in 20 μM ANS. Samples were equilibrated at room temperature in dark for ~10 min, ANS fluorescence was recorded between 400 – 550 nm by exciting the mixtures at 380 nm. Fluorescence of free ANS (blank) was subtracted from the sample readings.

2.8. Aggregation study

Aggregation of the purified rLdRab1 protein was induced by pH variation. 2 mg/ml protein solutions were prepared in buffers at pH 2.0 and pH 6.5 and incubated at 37° C. Kinetics of aggregation was monitored using ThT fluorescence as described earlier [24, 25]. Briefly, aggregation mixtures were diluted in 20 μM ThT to achieve final concentration of 2 μM. The protein samples were excited at 444 nm and the fluorescence emission was recorded between 460 - 550 nm. The Data are presented as ThT fluorescence at 485 nm as a function of time. The resulting plots were fitted with non-linear regression Eq. 3 to calculate kinetic parameters using equation 3.

where F₀ and F are the observed ThT fluorescence ratio at time zero and t minutes, respectively. t_1/2 is the time taken to reach 50% of maximum fluorescence. The apparent rate constant k_app is calculated as 1/ τ and lag time t_lag as ¼ t_1/2-2τ [24, 25].

2.9 Congo red (CR) assay

CR assay of final aggregates was performed as described earlier [24, 25]. Briefly, final aggregation reaction mixtures were diluted in 60 mM phosphate buffer pH 7.4 and 20 μM CR pH 7.4 to obtain protein concentration of 2 μM. Samples were allowed to equilibrate at room temperature for ~ 10 min. Absorption spectra were collected in the visible range between 400 nm – 700 nm. Difference absorption was calculated by subtracting spectrum of CR and respective aggregate from CR bound aggregate.

2.10 90⁰ light scattering (RLS)

Light scattering by aggregate particles was monitored by 90⁰ light scattering (RLS) [26] on Spectramax M2E spectrofluorometer (Molecular Devices). Final aggregation mixtures were diluted to obtain 2 μM of protein concentration in a phosphate buffer of pH 7.4. Samples were allowed to equilibrate at room temperature for ~ 10 min. RLS was recorded by setting the excitation and emission wavelengths at 350 nm each. Data are represented as RLS intensity normalised to fold increase with respect to control.

2.11 TEM imaging

The morphology of the final aggregation products was analysed using Transmission Electron Microscope (TEM, JEM-1400 Flash TEM). Aggregation reaction mixtures were diluted 3-fold (~0.8 mg/ml) with deionised water. 5 μl of diluted sample was layered on 200 mesh glow discharged carbon coated copper grid and adsorbed for 90 seconds. The grids were washed and stained with 2% phosphotungstic acid. Excess stains were washed and air dried. Images of the stained samples were taken on JEM-1400 Flash TEM operating at 120 kV.

3.1. The conserved nature of rLdRab1

This study utilizes molecular biology methods to express and purify the protein after cloning the LdRab1 sequence from the NCBI Ld database. The recombinant protein rLdRab1 has 222 amino acid residues, 55 of which are charged, and 6 of the histidines are at the N-terminus. Rab subfamily-specific structural components have been identified as conserved in all eukaryotic isoforms. [5]. Multiple sequence alignment of rLdRab1 performed using BLAST identified the small GTP and P-loop NTPase superfamily domain, the Rab1 family (RabF1-F5), Rab subfamily (RabSF2-SF4), GDI and GEF interaction elements, as defined for Rab proteins (Fig. S1) [5, 10].

The secondary structure prediction tools JPRED (https://www.compbio.dundee.ac.uk/jpred/), PredictProtein (https://predictprotein.org/), Phyre2 (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) estimate residue stretches 13-20, 48-58, 61-68, 89-95, 122-127, 153-157 to be participating in β strand formation; and 27-34, 73-84, 100-115, 140-149, 165-183 to compose helices (Fig. S2A). Homology alignments determined up to 90% similarity in the orientation and organization of beta and alpha stretches and an alpha-beta 3-layer (aba) sandwich at C end of rLdRab1 with lower protozoa like Cryptosporidium parvum (Fig. S2B) and up to 70 % with Rab 8A (2fu5) from Mus musculus (Fig. S2C) and YPT1 (1yzn) from Saccharomyces cerevisiae (Fig. S2D). A study elucidating the molecular functions of a Rab1 homologue from L. donovani reported up to ~80% similarity of the homologue with protozoan parasites, yeast and mice [8]. The sequence identity with rLdRab1 is low but there exists considerable structural similarity. Such homology between Rab isoforms has been previously reported. However, Rab1 isoforms in Leishmania genus is not previously discussed. This protein is implicated to function as a molecular switch to regulate vesicle trafficking pathways [7, 27].

The 3-D structure was modelled and analysed using SWISS Model (https://swissmodel.expasy.org/) and PHYRE2 (Fig. S3). The ribbon diagram generated depicts acidic residues in red and the basic residues in blue. The hydrophobic β strands occupy the buried core protein, whereas the helix and loop region are relatively solvent interactive. Such models of Rab1 have been elucidated and described. Globe predictions suggest rLdRab1 to be loose globular, lacking the typical compactness of a globular domain (table S2). A possible disulfide bonding is predicted between Cys 215, 216 (table S3).

3.2. pH induced unfolding is a non-two-state process

The solution behaviour of rLdRab1 isoform was predicted using sequence-based in silco analysis and biophysical techniques. Various in silico physico-chemical characteristics of rLdRab1 computed by ExPASy ProtParam (https://web.expasy.org/protparam/) are listed in table S1. The protein is slightly basic with a pI of 8.52. The aliphatic index which defines the relative volume occupied by the aliphatic side chain was computed to be 65.6. For thermostable proteins, the reported aliphatic index is garter than 90. This suggests rLdRab1 to be partially unstable or short-lived. As an index for protein hydrophobicity, Grand average of hydropathicity (GRAVY) was computed to be – 0.544. Proteins with a GRAVY score above 0 are more likely to be hydrophobic. This states the mild hydrophilic and polar nature of rLdRab1 isoform.

To understand the biophysical nature of rLdRab1 unfolding, the tertiary alterations induced on lowering the solution pH were monitored by intrinsic fluorescence at 340 nm and ANS fluorescence at 480 nm (Fig. 1A, B). IF and ANS assay effectively monitor the alterations in 3-D conformations on denaturation. Intrinsic fluorescence spectrum reflects the perturbation around the local interactions of Tyr and Trp residues. The fluorescence obtained by exciting the protein at 280 nm is due to tryptophan residues and energy transfer from the tyrosine residues, whereas the fluorescence obtained by the excitation at 297 nm is mainly due to tryptophan. Trp fluorescence is sensitive to the polarity of its micro-environment. Spectral properties such as λ_maxand intensity convey valuable information about the local solvent environment [28, 29]. rLdRab1 possess three tryptophanyl residues at position 146, 108 and 68 and two of the tyrosinyl residues at 13, 84 are predicted to have up to 25 % of solvent accessibility, whereas tyrosinyl placed at 40, 43 and 58 are relatively solvent interactive (Fig. S4).

The pH dependence of tryptophanyl fluorescence has been described earlier [30]. Signals of the fluorescence probes in the pH range 6 – 8 suggest stable native like conformations. A gradual decrease IF intensities accompanied by increase in ANS fluorescence is observed as pH is reduced from 5.5 to 3.0 (Fig. 1A-C). This corresponds to the unfolding transitions from native structures as ANS fluorescence is a direct estimate of surface exposed hydrophobic elements. Further decrease in pH saturates the lowest IF intensities and maximum ANS fluorescence (Fig. 1A-C). This illustrates accumulation of an unfolded intermediate below pH 3. The midpoint of transition from all three probes was calculated around pH 4 (Fig. 1D). However, differences in the slopes of unfolding (Fig. 2D) resulted in non-superimposable transitions (Fig. 1C, D). Hence there exists more than one transition intermediates during the acidic unfolding of rLdRab1. This intermediate which accumulates in the low pH range 1.5-3 demonstrates typical characteristics described for protein folding intermediates: an exposed hydrophobic core and loose structural arrangements [31, 32]. Similar observations have been described for a rLdRab5 and its mutants wherein urea induced unfolding accumulated at least three intermediate states [10].

3.3. The partially unfolded intermediate at pH 2

The rLdRab1 isoform purified at pH 6.5 was physically denatured at pH 2 and chemically in 6 M GuHCl. The content of secondary structure and tertiary conformations in different solution conditions were elucidated by far UV CD and fluorescence probes respectively (Fig. 2). The far UV CD spectrum of rLdRab1 isoform converted to MRE measured negative peaks at 209 nm and 222 nm at pH 6.5 (Fig. 2A). The negative ellipticity at 209 is dominant than that at 222 nm, which corresponds to the α+β structural class of proteins. The CD spectrum is significantly resembles to lysozyme, a model α+β protein [24]. The helical content of native rLdRab1 isoform was determined to be 38 % which is close to the estimated values of secondary structure prediction tools (32.41 %, 23.61 % and 43.98 %, respectively of helix, extended beta and loop structures and belonging to the α+β class). The isoform is almost unfolded in 6 M GuHCl, with only ~10 % of secondary structure (Fig. 2A). At pH 2, the negative ellipticity at 208 nm and 222 nm were observed to be similar in magnitude with a net 25 % loss in secondary structure (Fig. 2A). The tendency to adopt helix or beta transitions were estimated by ellipticity ratios (table 1). θ₂₀₅/θ₂₂₂was calculated for disorder changes,θ₂₁₆/θ₂₂₂ for beta structures and θ₂₂₂/θ₂₀₈for helical nature [33]. θ₂₂₂/θ₂₀₈at each pH was calculated to be < 1 which demonstrates a predominantly helical nature of the rLdRAb1 isoform at both pH (table 1). The marginal 4% increase in θ₂₁₅/θ₂₂₂ at pH 2 along with more than half the decrease in θ₂₀₄/θ₂₂₂corelates to a mild beta tendency following decreased in unstructured or disorder and helicity upon unfolding at pH 2 (table 1).

Table 1: Biophysical characterization of conformational states formed in pH 2, pH 6.5 and 6 M GuHCl.

Variables	pH 2	pH 6.5	6 M GuHCl
[θ]₂₂₂ (deg.cm².dmol^-1)	-8725.65	-11890.40	-1853.15
% helix	29.00 ± 0.14	37.96 ± 0.18	12.44 ± 0.05
[θ]₂₂₂/[θ]₂₀₈	1.09 ± 0.06	0.82 ± 0.01	-
[θ]₂₁₅/[θ]₂₂₂	1.11 ± 0.01	1.06 ± 0.06	2.16 ± 0.18
[θ]₂₀₄/[θ]₂₂₂	0.25 ± 0.05	0.98 ± 0.08
λ_max (nm) (IF)	340 ± 1.4	337 ± 1.3	352 ± 1.0
I₃₆₀/I₃₃₀(IF)	0.91	0.78	1.07
I₃₄₀ (IF)	601.72	1392.51	94.39
λ_max (nm) (TRP F)	339 ± 0.64	337 ± 0.52
I₃₆₀/I₃₃₀(TRP F)	0.92	0.79
I₃₄₀ (TRP F)	252.56	581.82
ANS FI 480 nm	1708.061	39.872	50.406

The IF spectra of rLDRab1 at pH 6.5 pH 2and exhibits λ_max in the 336-338 and 336-340 nm range (Fig. 2B). The pH 2 unfolded isoform records marginally broader λ_max with ~2 nm red shift and ~55 % quenched fluorescence (Fig.2B, table 1)_.Solvent interacting Trp residues have reported λ_maxof~345-350nm [29]. The isoform is almost fully denatured in 6 M GuHCl. ANS dye strongly interacts with exposed hydrophobic clusters, which significantly enhances fluorescence of the bound dye. ANS fluorescence directly corelates with exposed hydrophobic surfaces and efficiently monitors solvent exposure of hydrophobic clusters upon unfolding [34, 35]. ANS fluorescence of rLdRab1 at pH 6.5 and 6 M GuHCl overlap (Fig 2C), whereas there is ~12-fold increase at pH 2. This corresponds to perturbation of the core hydrophobic clusters, making their surface accessible for ANS binding. The results demonstrate the passage of acid-mediated denaturation of rLdRab1 through exposure of core hydrophobic clusters, retaining partial proportions of secondary and tertiary structure.

3.4. Aggregation kinetics

The phenomenon of protein aggregation describes the passage of soluble conformers generally partial or wrongly folded through a nucleation dependent mechanism to defined fibrillar aggregates [36]. Aggregation can proceed from either of the conformations that exist during protein folding, function, or degradations [37]. Since a partially unfolded intermediate accumulates at pH 2, its tendency to form amyloid aggregates was studied. The sequence of LdRab1 was studied for its amyloid behaviour in silico using AMYLPRED [38], which predicted stretches 14-23, 46-51, 70-83, 90-94 of LdRab1 sequence as aggregation prone. In vitro amyloid fibril formation of various model and diseased proteins have been described at pH 2 at various temperatures. [39, 40]. Time course of amyloid formation of 2 mg/ml rLdRab1 (~72 µM) at pH 2.0 and pH 6.5 at 310 K was followed by ThT fluorescence at 485 nm (Fig. 3). A sigmoidal increase in ThT fluorescence was observed at pH 2, which saturated after 5 days of incubation. To calculate parameters of aggregation kinetics, the data points was fitted with non-linear regression conveying a nucleation dependent model (Fig. 3). Lag time of ~ 68 h and half time of ~87 h was calculated. On the other hand, rLdRab1 at pH 6.5 did not record any significant ThT binding. After 72 h, precipitate like particles resulting in a turbid reaction mixture suggested amorphous aggregation (Fig. 3). The aggregation products present in the reaction mixtures after a period of 7 days were characterised by its tinctorial properties, secondary structure, and morphology.

3.5. Characterization of final aggregates

The morphology of the final aggregation products formed at pH 2.0 and pH 6.5 was examined up on negative staining using transmission electron microscopy (TEM) at a magnification of 60000X (Fig. 4). At pH 2, dense needle-like formations between 200 and 400 nm in length were observed (Fig. 4A). These structures resemble the protofibrils reported for various proteins in similar conditions [41–43]. At pH 6.5, the protein is converted into amorphous aggregates (Fig. 4B).

The secondary structures of aggregation products were determined by far-UV CD (Fig. 5). The protofibril formed at pH 2 (pH 2_a) showed a single broad negative peak spanning 212 – 219 nm, typically described for extended β structures (Fig. 5A) [25]. The aggregates at pH 6.5 (pH 6.5_a) record negative peaks at 209 nm and 222nm (Fig. 5A). There is a significant loss of CD signals as compared to native state at pH 6.5 (pH 6.5n). The structural transitions from native to the aggregated state is estimated from ellipticity ratios (Fig. 5B). Ellipticity ratio of θ₂₂₂/θ₂₀₈ of pH 6.5 aggregates (pH 6.5a) was calculated to be 1.1, suggesting dominance of helical nature in the aggregates. A decrease in disorder as well as beta content was observed. In protofibrils at pH 2, the decrease in disorder and increase in beta content is observed. The aggregates appear to retain helical nature of the recombinant protein.

Amyloid fibrils exhibit tinctorial (dye binding) properties which result in change in the spectral characteristics of the dyes [35]. The tinctorial characteristics of aggregates at pH 2 and pH 6.5 are described in Fig. 6 using ThT assay, CR difference spectra, ANS assay, and right-angle scattering (RLS). Fluorescence and difference spectroscopy of the dyes ThT and CR respectively, provide information of the content of β sheets as they specifically bind to cross β grooves [35, 44]. The protofibril-like aggregation products formed at pH 2 illustrate strong binding with ThT resulting in ~60% increase in ThT fluorescence (Fig. 6A) and a positive CR differential absorption (Fig. 6B). This demonstrates the presence and dominance of amyloid-like cross β sheet structures in the protofibrils. ~12-fold enhanced fluorescence ANS is observed in pH 2 formed aggregates (Fig. 6C) illustrating the significantly exposed hydrophobic clusters. Aggregation products of pH 6.5 do not present any tinctorial binding illustrating non amyloid, amorphous products. Light scattering (Fig. 6D) on the other hand provides information on particulate material present in the solution. Aggregates at pH 6.5 record ~50% higher RLS which determines its amorphous nature.

The vesicular cargo movement is coordinated by Rab proteins, which act as global molecular switches. In Leishmania, Rab functions are vital for the leishmaniasis infection and propagation [12]. Endocytic functions of Rab5 and Rab7 [10, 12] and secretory role of Rab1 [8] have been experimentally reported. Biophysical characteristics of a recombinant Rab5 have been described. However, information on structural aspects of LdRab1 is limited. This study aims to describe the solution characteristics of LdRab1. Using molecular biology techniques, we purified a recombinant Rab1 from L. donovani (rLdRab1) from the L. donovani database. Given the significant homology of rLdRab1 with the Rab1 proteins in protozoans and yeast but moderate identity with previously described LdRab1 homolog, we report here for the first time an isoform of Rab1 with implications in vesicular traffic regulation. Our in silico predictions and in vitro biophysical investigations are in good agreement with each other, that rLdRab1 belongs to the α+β structural class and is mildly polar in nature. The protein is predominantly helical in nature. As described for LdRab5, acid unfolding of rLdRab1 is a multistate process, which accumulates a partially unfolded intermediate. The conformational attributes of this intermediate characterised by significant native folds, exposed hydrophobic core and residual tertiary contacts; have been described for molten globule and pre-molten globule states. The molten globule posses’ significant secondary structure and globule-like compactness, parts of hydrophobic clusters but lacks native tertiary interactions. The pre-molten globule is less compact than the molten globule containing non-random structural elements and no tertiary structure. Both in-pathway folding intermediates are recognised as initiation points for amyloid aggregation.

The native state of a protein is one of the many conformations that a polypeptide polymer can achieve within a cell [37]. Apart from the native state, it is a generic behaviour of natural protein sequences to be predisposed to amyloid aggregation [36, 37]. Amyloids are described as insoluble fibrillar aggregates of soluble proteins, consisting of long protofilaments twisted to form a core cross β structure. The deposition of such amyloids have been identified as the pathological cause of many neurodegenerative and systemic disorders which are timely reviewed [36, 45]. Amyloidogenic segments have been identified for many disease related proteins [46]. In this work, we report and describe the amyloidogenic tendency of rLdRab1. We report amyloid like protofibrils of rLdRab1 at pH 2, whereas at pH 6.5 the amorphous nature of aggregation was observed. The existence of Rab protein in its different states are associated in human diseases including amyloid neuropathies and cancer [6, 27, 47, 48]. Our observations can have implication in targeting Rab proteins for Leishmania infections as well as amyloid

Herein, we cloned and purified a recombinant Rab1 protein from L donovani, rLdRab1 to elucidated its biophysical behaviour and amyloid nature. The protein was determined to be an isoform of LdRab1 protein implicated for vesicular traffic functions. Unfolding induced in an acidic environment was determined to be a multistate process using fluorescent probes. The characteristics of unfolding intermediates at low pH are traced intermedial of molten and pre-molten globule state. This intermediate is highly amyloidogenic and leads to the formation of protofibril-like structures. We report the aggregation predisposed behaviour of rLdRab1. Rab proteins have previously been hypothesised to be amyloidogenic, in vitro evidence of Rab amyloid like aggregation is less explored. Since the reasons for Rab related implications in Parkinson’s and Alzheimer’s disease are still unclear, our work can provide mechanistic approaches and opportunities for in depth studies on their correlation.

Acknowledgement

The authors gratefully acknowledge Department of MM and ME&T, Jamia Hamdard, New Delhi for laboratory facilities and financial assistance. Advanced Technology Platform Centre, Regional Centre for Biotechnology for TEM facility. Support of FIST (No. SR/FST/LS-I/2017/05[C]) grant to Department of Toxicology and PURSE grant (No. SR/PURSE Phase 2/39[C]) to Jamia Hamdard from Department of Science and Technology (DST), Ministry of Science and Technology, Govt. of India is acknowledged. B.A. gratefully acknowledges UGC-Faculty Recharge Program (UGC-FRP), UGC, Govt. of India for awarding UGC Assistant Professor position. SAM and RA acknowledge the ICMR-SRF position awarded by Indian Council of Medical Research (ICMR), Govt of India. AS acknowledges grant of Science and Engineering Research Board, Department of Science and Technology, India (EMR/2015/000874).

Competing interests

Authors have no competing interests to declare.

Author contributions

Roshanara: Data curation; Formal analysis; Investigation; Visualization; Methodology; Roles/Writing – original draft; Writing – review & editing

Shivani A. Muthu: Data curation; Formal analysis; Investigation; Visualization; Methodology; Roles/Writing – original draft; Writing – review & editing

Gulafsha: Data curation; Investigation; Visualization, Methodology

Rati Tandon: Data curation; Investigation; Visualization, Methodology

Angamuthu Selvapandiyan: Conceptualization; Funding acquisition; Project administration; Resources; Software; Supervision; Roles/Writing – original draft; Writing – review & editing.

Basir Ahmad: Conceptualization; Funding acquisition; Project administration; Resources; Software; Supervision; Roles/Writing – original draft; Writing – review & editing.

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Biophysical and Amyloid Behaviour of a Recombinant Rab1 Isoform of Leishmania Donovani

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