Neurofilament Light Protein Rod Domain Exhibits Structural Heterogeneity.

Neurofilaments are neuron-specific proteins that belong to the intermediate filament (IFs) protein family, with the neurofilament light chain protein (NFL) being the most abundant. The IFs structure typically includes a central coiled-coil rod domain comprised of coils 1A, 1B, and 2, separated by linker regions. The thermal stability of the IF molecule plays a crucial role in its ability for self-association. In the current study, we investigated the thermal stability of NFL coiled-coil domains by analyzing a set of recombinant domains and their fusions (NFL1B, NFL1A+1B, NFL2, NFL1B+2, and NFLROD) via circular dichroism spectroscopy and differential scanning calorimetry. The thermal stability of coiled-coil domains is evident in a wide range of temperatures, and thermal transition values (Tm) correspond well between isolated coiled-coil domains and full-length NFL. NFL1B has a Tm of 39.4 °C, and its' fusions, NFL1A+1B and NFL1B+2, have a Tm of 41.9 °C and 41.5 °C, respectively. However, in the case of NFL2, thermal denaturation includes at least two thermal transitions at 37.2 °C and 62.7 °C. These data indicate that the continuous α-helical structure of the coil 2 domain has parts with varied thermal stability. Among all the NFL fragments, only NFL2 underwent irreversible heat-induced denaturation. Together, these results unveil the origin of full-length NFL's thermal transitions, and reveal its domains structure and properties.

Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii.

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

Structural and Functional Properties of Tropomyosin Isoforms Tpm4.1 and Tpm2.1.

Tropomyosin (Tpm) is one of the most important partners of actin filament that largely determines its properties. In animal organisms, there are different isoforms of Tpm, which are believed to be involved in the regulation of various cellular functions. However, molecular mechanisms by which various Tpm cytoplasmic regulate of the functioning of actin filaments are still poorly understood. Here, we investigated the properties of Tpm2.1 and Tpm4.1 isoforms and compared them to each other and to more extensively studied Tpm isoforms. Tpm2.1 and Tpm4.1 were very similar in their affinity to F-actin, thermal stability, and resistance to limited proteolysis by trypsin, but differed markedly in the viscosity of their solutions and thermal stability of their complexes with F-actin. The main difference of Tpm2.1 and Tpm4.1 from other Tpm isoforms (e.g., Tpm1.6 and Tpm1.7) was their extremely low thermal stability as measured by the CD and DSC methods. We suggested the possible causes of this instability based on comparing the amino acid sequences of Tpm4.1 and Tpm2.1 with the sequences of Tpm1.6 and Tpm1.7 isoforms, respectively, that have similar exon structure.

Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation.

We characterized a novel genetic variant c.292G > A (p.E98K) in the TPM1 gene encoding cardiac tropomyosin 1.1 isoform (Tpm1.1), found in a proband with a phenotype of complex cardiomyopathy with conduction dysfunction and slow progressive neuromuscular involvement. To understand the molecular mechanism by which this mutation impairs cardiac function, we produced recombinant Tpm1.1 carrying an E98K substitution and studied how this substitution affects the structure of the Tpm1.1 molecule and its functional properties. The results showed that the E98K substitution in the N-terminal part of the Tpm molecule significantly destabilizes the C-terminal part of Tpm, thus indicating a long-distance destabilizing effect of the substitution on the Tpm coiled-coil structure. The E98K substitution did not noticeably affect Tpm's affinity for F-actin but significantly impaired Tpm's regulatory properties. It increased the Ca2+ sensitivity of the sliding velocity of regulated thin filaments over cardiac myosin in an in vitro motility assay and caused an incomplete block of the thin filament sliding at low Ca2+ concentrations. The incomplete motility block in the absence of Ca2+ can be explained by the loosening of the Tpm interaction with troponin I (TnI), thus increasing Tpm mobility on the surface of an actin filament that partially unlocks the myosin binding sites. This hypothesis is supported by the molecular dynamics (MD) simulation that showed that the E98 Tpm residue is involved in hydrogen bonding with the C-terminal part of TnI. Thus, the results allowed us to explain the mechanism by which the E98K Tpm mutation impairs sarcomeric function and myocardial relaxation.

Effect of Neurodegenerative Mutations in the NEFL Gene on Thermal Denaturation of the Neurofilament Light Chain Protein.

Effects of E90K, N98S, and A149V mutations in the light chain of neurofilaments (NFL) on the structure and thermal denaturation of the NFL molecule were investigated. By using circular dichroism spectroscopy, it was shown that these mutations did not lead to the changes in α-helical structure of NFL, but they caused noticeable effects on the stability of the molecule. We also identified calorimetric domains in the NFL structure by using differential scanning calorimetry. It was shown that the E90K replacement leads to the disappearance of the low-temperature thermal transition (domain 1). The mutations cause changes in the enthalpy of NFL domains melting, as well as lead to the significant changes in the melting temperatures (Tm) of some calorimetric domains. Thus, despite the fact that all these mutations are associated with the development of Charcot-Marie-Tooth neuropathy, and two of them are even located very close to each other in the coil 1A, they affect differently structure and stability of the NFL molecule.

Structural and Functional Properties of Kappa Tropomyosin.

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

Combined action of chemical chaperones on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules, which enhance protein stability, folding, inhibit protein aggregation, and are used for long-term storage of therapeutic proteins. The combined action of chemical chaperones trehalose, betaine and lysine on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b (Phb) has been studied. Dynamic light scattering data indicate that the affinity of trehalose to Phb increased in the presence of betaine or lysine at both stages (stage of nucleation and aggregate growth) of enzyme aggregation at 48 °C, in contrast, the affinity of betaine to the enzyme in the presence of lysine remained practically unchanged. According to differential scanning calorimetry and analytical ultracentrifugation data, the mixture of trehalose and betaine stabilized Phb stronger than either of them in total. Moreover, the destabilizing effect of lysine on the enzyme was almost completely compensated by trehalose and only partially by betaine. The main protective effect of the mixtures of osmolytes and lysine is associated with their influence on the dissociation/denaturation stage, which is the rate-limiting one of Phb aggregation. Thus, a pair of chaperones affects the stability, oligomeric state, and aggregation of Phb differently than individual chaperones.

De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), caused by mutations in thin filament proteins, manifests as moderate cardiac hypertrophy and is associated with sudden cardiac death (SCD). We identified a new de novo variant, c.656A>T (p.D219V), in the TPM1 gene encoding cardiac tropomyosin 1.1 (Tpm) in a young SCD victim with post-mortem-diagnosed HCM. We produced recombinant D219V Tpm1.1 and studied its structural and functional properties using various biochemical and biophysical methods. The D219V mutation did not affect the Tpm affinity for F-actin but increased the thermal stability of the Tpm molecule and Tpm-F-actin complex. The D219V mutation significantly increased the Ca2+ sensitivity of the sliding velocity of thin filaments over cardiac myosin in an in vitro motility assay and impaired the inhibition of the filament sliding at low Ca2+ concentration. The molecular dynamics (MD) simulation provided insight into a possible molecular mechanism of the effect of the mutation that is most likely a cause of the weakening of the Tpm interaction with actin in the "closed" state and so makes it an easier transition to the "open" state. The changes in the Ca2+ regulation of the actin-myosin interaction characteristic of genetic HCM suggest that the mutation is likely pathogenic.

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine.

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states-closed and open-in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Comparative structural and functional studies of low molecular weight tropomyosin isoforms, the TPM3 gene products.

Tropomyosin (Tpm) is an actin-associated protein and key regulator of actin filament structure and dynamics in muscle and non-muscle cells where it participates in many vital processes. Human non-muscle cells produce many Tpm isoforms; however, little is known yet about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five low molecular weight Tpm isoforms (Tpm3.1, Tpm3.2, Tpm3.4, Tpm3.5, and Tpm3.7), the products of TPM3 gene, which significantly differ by alternatively spliced internal exon 6 (6a or 6b) and C-terminal exon 9 (9a, 9c or 9d). Our results clearly demonstrate that the properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. These differences can be important in further studies to explain why these Tpm isoforms play a key role in organization and dynamics of the cytoskeleton.

Tropomyosin pseudo-phosphorylation can rescue the effects of cardiomyopathy-associated mutations.

We applied various methods to investigate how mutations S283D and S61D that mimic phosphorylation of tropomyosin (Tpm) affect structural and functional properties of cardiac Tpm carrying cardiomyopathy-associated mutations in different parts of its molecule. Using differential scanning calorimetry and molecular dynamics, we have shown that the S61D mutation (but not the S283 mutation) causes significant destabilization of the N-terminal part of the Tpm molecule independently of the absence or presence of cardiomyopathy-associated mutations. Our results obtained by cosedimentation of Tpm with F-actin demonstrated that both S283D and S61D mutations can reduce or even eliminate undesirable changes in Tpm affinity for F-actin caused by some cardiomyopathy-associated mutations. The results indicate that Tpm pseudo-phosphorylation by mutations S283D or S61D can rescue the effects of mutations in the TPM1 gene encoding a cardiac isoform of Tpm that lead to the development of such severe inherited heart diseases as hypertrophic or dilated cardiomyopathies.

Effects of myopathy-causing mutations R91P and R245G in the TPM3 gene on structural and functional properties of slow skeletal muscle tropomyosin.

Tropomyosin (Tpm) is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Numerous point mutations in the TPM3 gene encoding Tpm of slow skeletal muscles (Tpm 3.12 or γ-Tpm) are associated with the genesis of various congenital myopathies. Two of these mutations, R91P and R245G, are associated with congenital fiber-type disproportion (CFTD) characterized by hypotonia and generalized muscle weakness. We applied various methods to investigate how these mutations affect the structural and functional properties of γγ-Tpm homodimers. The results show that both these mutations lead to strong structural changes in the γγ-Tpm molecule and significantly impaired its functional properties. These changes in the Tpm properties caused by R91P and R245G mutations give insight into the molecular mechanism of the CFTD development and the weakness of slow skeletal muscles observed in this inherited disease.

Effect of arginine on stability and aggregation of muscle glycogen phosphorylase b.

Arginine (Arg) is frequently used in biotechnology and pharmaceutics to stabilize protein preparations. When using charged ions like Arg, it is necessary to take into account their contribution to the increase in ionic strength, in addition to the effect of Arg on particular processes occurring under the conditions of constancy of ionic strength. Here, we examined contribution of ionic strength (0.15 and 0.5 M) to the effects of Arg on denaturation, thermal inactivation and aggregation of skeletal muscle glycogen phosphorylase b (Phb). Dynamic light scattering, analytical ultracentrifugation, differential scanning calorimetry, circular dichroism and enzymatic activity assay were used to assess the effects of Arg at constant ionic strength compared with the effects of ionic strength alone. We found that high ionic strength did not affect the secondary structure of Phb, but changed conformation of the protein. Such a destabilization of the enzyme causes an increase in the initial rate of aggregation and inactivation of Phb thereby affecting its denaturation. Binding of Arg causes additional changes in the protein conformation, weakening the bonds between monomers in the dimer. This causes the dimer to dissociate into monomers, which rapidly aggregate. Thus, Arg acts on these processes much stronger than just ionic strength.

Mechanisms of disturbance of the contractile function of slow skeletal muscles induced by myopathic mutations in the tropomyosin TPM3 gene.

Several congenital myopathies of slow skeletal muscles are associated with mutations in the tropomyosin (Tpm) TPM3 gene. Tropomyosin is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Two Tpm isoforms, γ (Tpm3.12) and ß (Tpm2.2) are expressed in human slow skeletal muscles forming γγ-homodimers and γß-heterodimers of Tpm molecules. We applied various methods to investigate how myopathy-causing mutations M9R, E151A, and K169E in the Tpm γ-chain modify the structure-functional properties of Tpm dimers, and how this affects the muscle functioning. The results show that the features of γγ-Tpm and γß-Tpm with substitutions in the Tpm γ-chain vary significantly. The characteristics of the γγ-Tpm depend on whether these mutations located in only one or both γ-chains. The mechanism of the development of nemaline myopathy associated with the M9R mutation was revealed. At the molecular level, a cause-and-effect relationship has been established for the development of myopathy by the K169E mutation. Also, we described the structure-functional properties of the Tpm dimers with the E151A mutation, which explain muscle weakness linked to this substitution. The results demonstrate a diversity of the molecular mechanisms of myopathy pathogenesis induced by studied Tpm mutations.

Thermal unfolding of various human non-muscle isoforms of tropomyosin.

Tropomyosin (Tpm) is an α-helical coiled-coil protein dimer, which forms a continuous head-to-tail polymer along the actin filament. In striated muscles, Tpm plays an important role in the Ca2+-dependent regulation of muscle contraction. However, little is known about functional and especially structural properties of the numerous non-muscle Tpm isoforms. In the present work, we have applied circular dichroism (CD) and differential scanning calorimetry (DSC) to investigate thermal unfolding and domain structure of various non-muscle human Tpm isoforms. These isoforms, the products of two different genes, TPM1 and TPM3, also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results clearly demonstrate that structural properties of various non-muscle Tpm isoforms can be quite different depending on the presence of different alternatively spliced exons in their genes. These data show for the first time a significant difference in the thermal unfolding between muscle and non-muscle Tpm isoforms and indicate that replacement of alternatively spliced exons alters the stability of certain domains in the Tpm molecule.

Structural and functional properties of αß-heterodimers of tropomyosin with myopathic mutations Q147P and K49del in the ß-chain.

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca2+-regulated contraction of striated muscles. Two Tpm isoforms, α (Tpm 1.1) and ß (Tpm 2.2), are expressed in fast skeletal muscles. These Tpm isoforms can form either αα and ßß homodimers, or αß heterodimers. However, only αα-Tpm and αß-Tpm dimers are usually present in most of fast skeletal muscles, because ßß-homodimers are relatively unstable and cannot exist under physiologic conditions. Nevertheless, the most of previous studies of myopathy-causing mutations in the Tpm ß-chains were performed on the ßß-homodimers. In the present work, we applied different methods to investigate the effects of two myopathic mutations in the ß-chain, Q147P and K49del (i.e. deletion of Lys49), on structural and functional properties of Tpm αß-heterodimers and to compare them with the properties of ßß-homodimers carrying these mutations in both ß-chains. The results show that the properties of αß-Tpm heterodimers with these mutations in the ß-chain differ significantly from the properties of ßß-homodimers with the same substitutions in both ß-chains. This indicates that the αß-heterodimer is a more appropriate model for studying the effects of myopathic mutations in the ß-chain of Tpm than the ßß-homodimer which virtually does not exist in human skeletal muscles.

Thermal unfolding of homodimers and heterodimers of different skeletal-muscle isoforms of tropomyosin.

We applied differential scanning calorimetry (DSC) to investigate the structural properties of three isoforms of tropomyosin (Tpm), α, ß, and γ, expressed from different genes in human skeletal muscles. We compared specific features of the thermal unfolding of αα, ßß, and γγ Tpm homodimers, as well as of αß and Î³ß Tpm heterodimers. The results show that the thermal stability of γγ homodimer is much higher than that of αα homodimer which, in turn, is much more thermostable than the ßß homodimer. The stability of the Î³ß Tpm heterodimer is much lower than that of the γγ homodimer, and its thermal unfolding is quite different from that for γγ and ßß homodimers, whereas the unfolding of the αß heterodimer is roughly similar to that of the αα homodimer.

Structural basis of the high thermal stability of the histone-like HU protein from the mollicute Spiroplasma melliferum KC3.

The three-dimensional structure of the histone-like HU protein from the mycoplasma Spiroplasma melliferum KC3 (HUSpm) was determined at 1.4 Å resolution, and the thermal stability of the protein was evaluated by differential scanning calorimetry. A detailed analysis revealed that the three-dimensional structure of the HUSpm dimer is similar to that of its bacterial homologues but is characterized by stronger hydrophobic interactions at the dimer interface. This HUSpm dimer interface lacks salt bridges but is stabilized by a larger number of hydrogen bonds. According to the DSC data, HUSpm has a high denaturation temperature, comparable to that of HU proteins from thermophilic bacteria. To elucidate the structural basis of HUSpm thermal stability, we identified amino acid residues potentially responsible for this property and modified them by site-directed mutagenesis. A comparative analysis of the melting curves of mutant and wild-type HUSpm revealed the motifs that play a key role in protein thermal stability: non-conserved phenylalanine residues in the hydrophobic core, an additional hydrophobic loop at the N-terminal region of the protein, the absence of the internal cavity present at the dimer interface of some HU proteins, and the presence of additional hydrogen bonds between the monomers that are missing in homologous proteins.

Effect of GroEL on thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

The suppression of the thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle by the chaperonin GroEL is studied using dynamic light scattering. It is shown that the decrease in the rate of Phb aggregation under the action of GroEL is due to the transition of the aggregation process from the kinetic regime, wherein the rate of aggregation is limited by diffusion of the interacting particles, to a regime where the sticking probability for the colliding particles becomes lower than one (reaction-limited cluster-cluster aggregation). The analytical-ultracentrifugation data show that elevated temperatures induce dissociation of the dimeric Phb. The formation of a complex between the denatured monomeric form of Phb and the dissociated forms of GroEL is detected during heating at 46 degrees C.