Dynein Light Chain Protein Research Articles

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Loss of Axdnd1 causes sterility due to impaired spermatid differentiation in mice.

PurposeSpermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm motility. Here, we focused on the gene axonemal dynein light chain domain containing 1 (Axdnd1) with the aim to determine the function of its protein product AXDND1.MethodsTo elucidate the role of AXDND1 in spermatogenesis, we generated Axdnd1 knockout (KO) mice using the CRISPR/Cas9 system. The generated mice were subjected to fertility tests and analyzed by immunohistochemistry.ResultThe Axdnd1 KO mouse exhibited sterility caused by impaired spermiogenesis during the elongation step as well as abnormal nuclear shaping and manchette, which are essential for spermiogenesis. Moreover, AXDND1 showed enriched testicular expression and was localized from the mid‐pachytene spermatocytes to the early spermatids.Conclusion Axdnd1 is essential for spermatogenesis in the mouse testes. These findings improve our understanding of spermiogenesis and related defects. According to a recent report, deleterious heterozygous mutations in AXDND1 were found in non‐obstructive azoospermia (NOA) patients. Therefore, Axdnd1 KO mice could be used as a model system for NOA, which will greatly contribute to future NOA treatment studies.

The dynein light chain protein Tda2 functions as a dimerization engine to regulate actin capping protein during endocytosis

Clathrin- and actin-mediated endocytosis is a fundamental process in eukaryotic cells. Previously, we discovered Tda2 as a new yeast dynein light chain (DLC) that works with Aim21 to regulate actin assembly during endocytosis. Here we show Tda2 functions as a dimerization engine bringing two Aim21 molecules together using a novel binding surface different than the canonical DLC ligand binding groove. Point mutations on either protein that diminish the Tda2-Aim21 interaction in vitro cause the same in vivo phenotype as TDA2 deletion showing reduced actin capping protein (CP) recruitment and increased filamentous actin at endocytic sites. Remarkably, chemically induced dimerization of Aim21 rescues the endocytic phenotype of TDA2 deletion. We also uncovered a CP interacting motif in Aim21, expanding its function to a fundamental cellular pathway and showing such motif exists outside mammalian cells. Furthermore, specific disruption of this motif causes the same deficit of actin CP recruitment and increased filamentous actin at endocytic sites as AIM21 deletion. Thus, the data indicate the Tda2-Aim21 complex functions in actin assembly primarily through CP regulation. Collectively, our results provide a mechanistic view of the Tda2-Aim21 complex and its function in actin network regulation at endocytic sites.

Molecular principles of Piwi-mediated cotranscriptional silencing through the dimeric SFiNX complex.

Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.

A tomato dynein light chain gene SlLC6D is a negative regulator of chilling stress

Dynein light chain (DLC) proteins are an important component of dynein complexes, which are widely distributed in plants and animals and involved in a variety of cellular processes. The functions of DLC genes in plant chilling stress remain unclear. In this study, we isolated a DLC gene from tomato, designated SlLC6D. Promoter analysis revealed many cis-elements involved in abiotic stress in the SlLC6D promoter. Expression of SlLC6D was induced by heat and salt stress, and inhibited by polyethylene glycol and chilling stress. Knockdown of SlLC6D in tomato exhibited low relative electrolyte leakage, malondialdehyde content, and reactive oxygen species (ROS) accumulation under chilling stress. The content of proline and activities of superoxide dismutase and peroxidase in knockdown lines were higher than in the wild type and overexpression lines during chilling stress. The high transcript abundances of three cold-responsive genes were detected in knockdown lines in response to chilling stress. Seedling growth of knockdown lines was significantly higher than that of the wild type and overexpression lines under chilling stress. These results suggest that SlLC6D is a negative regulator of chilling stress tolerance, possibly by regulating ROS contents and the ICE1–CBF–COR pathway.

PI31 Is an Adaptor Protein for Proteasome Transport in Axons and Required for Synaptic Development

Protein degradation by the ubiquitin-proteasome system is critical for neuronal function. Neurons utilize microtubule-dependent molecular motors to allocate proteasomes to synapses, but how proteasomes are coupled to motors and how this is regulated to meet changing demand for protein breakdown remain largely unknown. We show that the conserved proteasome-binding protein PI31 serves as an adaptor to couple proteasomes with dynein light chain proteins (DYNLL1/2). The inactivation of PI31 inhibited proteasome motility in axons and disrupted synaptic proteostasis, structure, and function. Moreover, phosphorylation of PI31 by p38 MAPK enhanced binding to DYNLL1/2 and promoted the directional movement of proteasomes in axons, suggesting a mechanism to regulate loading of proteasomes onto motors. Inactivation of PI31 in mouse neurons attenuated proteasome movement in axons, indicating this process is conserved. Because mutations affecting PI31 activity are associated with human neurodegenerative diseases, impairment of PI31-mediated axonal transport of proteasomes may contribute to these disorders.

Tctex-1, a novel interaction partner of Kidney Injury Molecule-1, is required for efferocytosis.

Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is specifically upregulated on proximal tubular epithelial cells (PTECs) during acute kidney injury and mitigates tissue damage by mediating efferocytosis (the phagocytic clearance of apoptotic cells). The signaling molecules that regulate efferocytosis in TECs are not well understood. Using a yeast two-hybrid screen, we identified the dynein light chain protein, Tctex-1, as a novel KIM-1-interacting protein. Immunoprecipitation and confocal imaging studies suggested that Tctex-1 associates with KIM-1 in cells at baseline, but, dissociates from KIM-1 within 90 min of initiation of efferocytosis. Interfering with actin or microtubule polymerization interestingly prevented the dissociation of KIM-1 from Tctex-1. Moreover, the subcellular localization of Tctex-1 changed from being microtubule-associated to mainly cytosolic upon expression of KIM-1. Short hairpin RNA-mediated silencing of endogenous Tctex-1 in cells significantly inhibited efferocytosis to levels comparable to that of knock down of KIM-1 in the same cells. Importantly, Tctex-1 was not involved in the delivery of KIM-1 to the cell-surface. On the other hand, KIM-1 expression significantly inhibited the phosphorylation of Tctex-1 at threonine 94 (T94), a post-translational modification which is known to disrupt the binding of Tctex-1 to dynein on microtubules. In keeping with this, we found that KIM-1 bound less efficiently to the phosphomimic (T94E) mutant of Tctex-1 compared to wild type Tctex-1. Surprisingly, expression of Tctex-1 T94E did not influence KIM-1-mediated efferocytosis. Our studies uncover a previously unknown role for Tctex-1 in KIM-1-dependent efferocytosis in epithelial cells.

Desmosomal cadherin association with a dynein‐cortactin complex promotes actin rearrangements required for epidermal morphogenesis

The epidermis is a multi‐layered, renewing epithelium that serves as an essential barrier against water loss and environmental insults in vertebrates. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes in which epidermal keratinocytes in the basal layer commit to differentiate, and move towards the skin's surface in a process called stratification. During this process, tissue integrity is ensured by intercellular junctions called desmosomes, which anchor the intermediate filament cytoskeleton to sites of desmosomal cadherin‐mediated adhesion. In contrast to the adherens junction protein, E‐cadherin, which is expressed in all epidermal layers, seven desmosomal cadherins are expressed in a highly patterned differentiation‐dependent manner in epidermis. This genomic complexity and spatial patterning of desmosome components in stratified tissues suggests that they do more than perform textbook functions in mediating cell‐cell adhesion. Here, we reveal an unexpected role for the cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into suprabasal layers through a process of delamination/extrusion. This function requires a newly recognized association between Dsg1 and the dynein light chain, Tctex‐1, and actin scaffolding protein, cortactin. We demonstrate that Tctex‐1, as a part of the dynein complex, controls the proper biochemical compartmentalization and segregation of Dsg1‐containing desmosomes from adherens junctions. Moreover, uncoupling the Dsg1‐Tctex‐1 complex from dynein or abrogation of Dsg1‐Tctex‐1 interactions interfered with the proper positioning of cortactin, which in turn is required to recruit the Arp2/3 complex and promote F‐actin network reorganization. Assembly of this molecular complex at the onset of Dsg1 expression during differentiation in cells already containing E‐cadherin results in decreased tension at adherens junctions, as measured through an E‐cadherin FRET sensor and accumulation of the mechanosensitive molecule vinculin. The proper coordination of this chain of events is required for the efficient delamination of basal cells in an organotypic model of tissue development. Thus, Dsg1 serves as a scaffold that exerts spatial control over mechanical changes necessary to promote early stages of epidermal morphogenesis.Support or Funding InformationSupported by NIH R01AR041836, R37AR043380, R01CA122151, the Dermatology Foundation and J.L. Mayberry Endowment.

Dynein light chain family genes in 15 plant species: Identification, evolution and expression profiles

Dynein light chain (DLC) is one important component of the dynein complexes, which have been proved involving in a variety of cellular functions. However, higher plants lack all other components of the complexes except DLCs, suggesting that in plants, the DLC protein does not carry out the same function as it in animals. Therefore, the function of this family in plants is mysterious. In this study, we investigated the DLC gene family in 15 plant species and analyzed their expression profiles. In total, 128 DLC genes were identified from the 15 studied plant species and were divided into eight groups by their phylogenetic relation. Highly conserved gene structure and motif arrangement was discovered within each group, indicating their functional correlation. Genetic variation and recombination events were also detected in DLC genes. Through selection analyses, we also identified some significant site-specific constraints in most of the DLC paralogs. In addition, DLC genes presented various expression profiles in different development stages, or under different abiotic stresses or phytohormone treatments. This may be associated with a variety of cis-elements responding to stress and phytohormone in the upstream sequences of the DLC genes. Functional network analysis exhibited 123 physical or functional interactions. The results provide a foundation for exploring the characterization of the DLC genes in plants and offer insights for additional functional studies.

Pex17p-dependent assembly of Pex14p/Dyn2p-subcomplexes of the peroxisomal protein import machinery

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by peroxisomal targeting sequences (PTS). In the following, the assembled receptor-cargo complex is targeted to the peroxisomal membrane where it docks to the docking-complex as part of the peroxisomal translocation machinery. The docking-complex is composed of Pex13p, Pex14p and in yeast also Pex17p, whose function is still elusive. In order to characterize the function of Pex17p, we compared the composition and size of peroxisomal receptor-docking complexes from wild-type and pex17Δ cells. Our data demonstrate that the deficiency of Pex17p affects the stoichiometry of the constituents of an isolated 600kDa complex and that pex17Δ cells lack a high molecular weight complex (>900kDa) of unknown function. We identified the dynein light chain protein Dyn2p as an additional core component of the Pex14p/Pex17p-complex. Both, Pex14p and Pex17p interact directly with Dyn2p, but in vivo, Pex17p turned out to be prerequisite for an association of Dyn2p with Pex14p. Finally, like pex17Δ also dyn2Δ cells lack the high molecular weight complex. As dyn2Δ cells also display reduced peroxisomal function, our data indicate that Dyn2p-dependent formation of the high molecular weight Pex14p-complex is required to maintain peroxisomal function on wild-type level.

Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate

Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications.

Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy.

We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear (1)H-X (X = (13)C, (15)N, (31)P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the (1)H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the (1)H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from (1)H chemical shift anisotropy, while keeping the (1)H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [(15)N]-N-acetyl-valine and [U-(13)C,(15)N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate (1)H-(15)N dipolar couplings in the context of 3D experiments is presented on U-(13)C,(15)N-enriched dynein light chain protein LC8.

BIM doubles its responsibilities

The pro-apoptotic BH3-only protein BIM is inactivated by binding to the dynein light chain protein LC8. However, under conditions of stress BIM is phosphorylated by Jun N-terminal kinase (JNK), which leads to its dissociation from LC8 and localization at mitochondria, where it can induce apoptosis. This study finds that the interaction of BIM with LC8 also regulates autophagy. Under steady-state conditions, LC8 mediates binding of apoptosis-inactive BIM to BECLIN1, leading to the mislocalization of the latter from the endoplasmic reticulum to microtubules, where it cannot promote autophagosome formation and maturation. However, under starvation conditions BIM is phosphorylated by JNK, thereby releasing BECLIN1 and allowing autophagy induction. Consistent with this, loss of BIM leads to enhanced autophagosome induction. Thus, similarly to anti-apoptotic BH3-only proteins, BIM has a key role in inhibiting autophagy under steady-state conditions.

Mechanistic Insight into the Microtubule and Actin Cytoskeleton Coupling through Dynein-Dependent RhoGEF Inhibition

Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.

Residual structure and dynamics in DMSO-d6 denatured Dynein Light Chain protein

Structural and motional features in the denatured state of a protein dictate the early folding events starting from that state and these features vary depending upon the nature of the denaturant used. Here, we have attempted to decipher the early events in the folding of Dynein Light Chain protein (DLC8), starting from DMSO-d6 denatured state. Multinuclear NMR experiments were used to obtain the full spectral assignment. The HSQC spectrum shows the presence of two sets of peaks for the residues Met 1, Ser 2, Arg 4, Ala 11, Met 17, Thr 26, Lys 44, Tyr 50, Asn 51, Trp 54, His 55, Val 58, Gly 59, Ser 64, Tyr 65, His 68, Phe 86, Lys 87 indicating the presence of slow conformational transition in the heterogeneous ensemble. Analysis of residual structural propensities with secondary 13C chemical shifts, 3J(HN−Hα) coupling constants and 1H-1H NOE revealed the presence of local preferences which encompass both native and non-native like structures. The spectral density calculations, as obtained from measured R1, R2 and 1H-15N steady state NOE values provide insights into the backbone dynamics on the milli to picosecond timescale. The segment Ser 14 – His 55 exhibits slow motions on the milli- to microsecond timescale arising from conformational exchange. The presence of native like structural preference, as well as conformational exchange classifies the above segment as the nucleation site of folding. Based on the observations, we propose here, the probable hierarchy of folding of DLC8 on dilution of denaturant: the two helices are formed first followed by the formation of β2 and β5.

An Ana2/Ctp/Mud Complex Regulates Spindle Orientation in Drosophila Neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function toorient the mitotic spindle during NB asymmetric division.

Hierarchy of local structural and dynamics perturbations due to subdenaturing urea in the native state ensemble of DLC8 dimer

Local structural and dynamic modulations due to small environmental perturbations reflect the adaptability of the protein to different interactors. We have investigated here the preferential local perturbations in Dynein light chain protein (DLC8), a cargo adapter, by sub-denaturing urea concentrations. Equilibrium unfolding experiments by optical spectroscopic methods indicated a two state like unfolding of DLC8 dimer, with the transition mid-point occurring around 8.6 M urea. NMR studies identified the β3 and β4 strands, N-, C- terminal regions, loops connecting β1 to α1, α1 to α2 and β3 to β4 as the soft targets of urea perturbation and thus indicated potential unfolding initiation sites. Native-state hydrogen exchange studies suggested the unfolding to traverse from the edges towards the centre of the secondary structural elements. At 6 M urea the whole protein chain acts like a cooperative unit. These observations are expected to have important implications for the protein's multiple functions.

Structure-function-folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure-function-folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years.

HIV‐1 integrase trafficking in S. cerevisiae: a useful model to dissect the microtubule network involvement of viral protein nuclear import

Intracellular transport of karyophilic cargos comprises translocation to the nuclear envelope and subsequent nuclear import. Small cargos such as isolated proteins can reach the nuclear envelope by diffusion but movement of larger structures depends on active translocation, typically using microtubules. Centripetal transport ends at the perinuclear microtubule organizing centre called the spindle pole body (SPB) in yeast. Previously, we found by two hybrids that the karyophilic lentiviral-encoded integrase (IN) interacts with two yeast microtubule-associated proteins, Dyn2p (dynein light chain protein) and Stu2p, a centrosomal protein (de Soultrait et al., 2002). Thus, to investigate the hinge between cytoplasmic retrograde transport and nuclear import, we decided to analyse HIV-1 IN trafficking in yeast as the model, since each of these biological mechanisms is evolutionarily conserved in eukaryotic cells. Here, we found an accumulation of IN at the SPB in yeast via Stu2p colocalization. Disruption of the microtubule network by nocodazole or IN expression in a dynein 2-deficient yeast strain prevented IN accumulation in the nuclear periphery and additionally inhibited IN transport into the nucleus. By mutagenesis, we showed that trafficking of IN towards the SPB requires the C-terminus of the molecule. Taking our findings together, we proposed a model in which IN nuclear import seems to depend on an essential intermediate step in the SPB. We found that Dyn2p and Stu2p play an important role in driving IN toward MTOC and could optimize nuclear entry of the retroviral enzyme. Our results suggest a new hypothesis in keeping with the current HIV-1 intracellular trafficking model.

PH dependent unfolding characteristics of DLC8 dimer: Residue level details from NMR

Environment dependence of folding and unfolding of a protein is central to its function. In the same vein, knowledge of pH dependence of stability and folding/unfolding is crucial for many biophysical equilibrium and kinetic studies designed to understand protein folding mechanisms. In the present study we investigated the guanidine induced unfolding transition of dynein light chain protein (DLC8), a cargo adaptor of the dynein complex in the pH range 7–10. It is observed that while the protein remains a dimer in the entire pH range, its stability is somewhat reduced at alkaline pH. Global unfolding features monitored using fluorescence spectroscopy revealed that the unfolding transition of DLC8 at pH 7 is best described by a three-state model, whereas, that at pH 10 is best described by a two-state model. Chemical shift perturbations due to pH change provided insights into the corresponding residue level structural perturbations in the DLC8 dimer. Likewise, backbone 15N relaxation measurements threw light on the corresponding motional changes in the dimeric protein. These observations have been rationalized on the basis of expected changes with increasing pH in the protonation states of the titratable residues on the structure of the protein. These, in turn provide an explanation for the change from three-state to two-state guanidine induced unfolding transition as the pH is increased from 7 to 10. All these results exemplify and highlight the role of environment vis-à-vis the sequence and structure of a given protein in dictating its folding/unfolding characteristics.