Arginine-binding Protein Research Articles

Destabilization of the D2 domain of Thermotoga maritima arginine binding protein induced by guanidinium thiocyanate and its counteraction by stabilizing agents.

D2 is a structural and cooperative domain of Thermotoga maritima Arginine Binding Protein, that possesses a remarkable conformational stability, with a denaturation temperature of 102.6°C, at pH 7.4. The addition of potassium thiocyanate causes a significant decrease in the D2 denaturation temperature. The interactions of thiocyanate ions with D2 have been studied by means of isothermal titration calorimetry measurements and molecular dynamics simulations. It emerged that: (a) 20-30 thiocyanate ions interact with the D2 surface and are present in its first solvation shell; (b) each of them makes several contacts with protein groups, both polar and nonpolar ones. The addition of guanidinium thiocyanate causes a marked destabilization of the D2 native state, because both the ions are denaturing agents. However, on adding to the solution containing D2 and guanidinium thiocyanate a stabilizing agent, such as TMAO, sucrose or sodium sulfate, a significant increase in denaturation temperature occurs. The present results confirm that counteraction is a general phenomenon for globular proteins.

The role of C-terminal helix in the conformational transition of an arginine binding protein

The thermotoga maritima arginine binding protein (TmArgBP) is a periplasmic binding protein that has a short helix at the C-terminal end (CTH), which is swapped between the two chains. We apply a coarse-grained structure-based model (SBM) and all-atom MD simulation on this protein to understand the mechanism and the role of CTH in the conformational transition. When the results of SBM simulations of TmArgBP in the presence and absence of CTH are compared, we find that CTH is strategically located at the back of the binding pocket restraining the open-state conformation thereby disengaging access to the closed-state. We also ran all-atom MD simulations of open-state TmArgBP with and without CTH and discovered that in the absence of CTH the protein could reach the closed-state within 250 ns, while in its presence, the protein remained predominantly in its open-state conformation. In the simulation started from unliganded closed-state conformation without CTH, the protein exhibited multiple transitions between the two states, suggesting CTH as an essential structural element to stabilize the open-state conformation. In another simulation that began with an unliganded closed-state conformation with CTH, the protein was able to access the open-state. In this simulation the CTH was observed to reorient itself to interact with the protein emphasizing its role in assisting the conformational change. Based on our findings, we believe that CTH not only acts as a structural element that constraints the protein in its open-state but it may also guide the protein back to its open-state conformation upon ligand unbinding.

The Role of C-Terminal Helix in the Conformational Transition of an Arginine Binding Protein

The Role of C-Terminal Helix in the Conformational Transition of an Arginine Binding Protein

The Role of C-Terminal Helix in the Conformational Transition of an Arginine Binding Protein

The Role of C-Terminal Helix in the Conformational Transition of an Arginine Binding Protein

Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima.

Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors.

Guanidinium binding to proteins: The intriguing effects on the D1 and D2 domains of Thermotoga maritima Arginine Binding Protein and a comprehensive analysis of the Protein Data Bank

Thermotoga maritima Arginine Binding Protein has been extensively characterized because of its peculiar features and its possible use as a biosensor. In this characterization, deletion of the C-terminal helix to obtain the monomeric protein TmArgBP20–233 and dissection of the monomer in its two domains, D1 and D2, have been performed. In the present study the stability of these three forms against guanidinium chloride is investigated by means of circular dichroism and differential scanning calorimetry measurements. All three proteins show a high conformational stability; moreover, D1 shows an unusual behavior in the presence of low concentrations of guanidinium chloride. This finding has led us to investigate a possible binding interaction by means of isothermal titration calorimetry and X-ray crystallography; the results indicate that D1 is able to bind the guanidinium ion (GuH+), due to its similarity with the arginine terminal moiety. The analysis of the structural and dynamic properties of the D1-GuH+ complex indicates that the protein binds the ligand through multiple and diversified interactions. An exhaustive survey of the binding modes of GuH+ to proteins indicates that this is a rather common feature. These observations provide interesting insights into the effects that GuH+ is able to induce in protein structures.

On the extraordinary pressure stability of the Thermotoga maritima arginine binding protein and its folded fragments - a high-pressure FTIR spectroscopy study.

The arginine binding protein from T. maritima (ArgBP) exhibits several distinctive biophysical and structural properties. Here we show that ArgBP is also endowed with a ramarkable pressure stability as it undergoes minor structural changes only, even at 10 kbar. A similar stability is also observed for its folded fragments (truncated monomer and individual domains). A survey of literature data on the pressure stability of proteins highlights the uncommon behavior of ArgBP.

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima.

Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant hasbeen structurally characterized. Analysis of this arginine-bound structure indicates that it consists of a closed monomer with its C-terminal helix folded against the rest of the protein, as typically observed for substrate-binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.

The characterization of Thermotoga maritima Arginine Binding Protein variants demonstrates that minimal local strains have an important impact on protein stability

The Ramachandran plot is a versatile and valuable tool that provides fundamental information for protein structure determination, prediction, and validation. The structural/thermodynamic effects produced by forcing a residue to adopt a conformation predicted to be forbidden were here explored using Thermotoga maritima Arginine Binding Protein (TmArgBP) as model. Specifically, we mutated TmArgBP Gly52 that assumes a conformation believed to be strictly disallowed for non-Gly residues. Surprisingly, the crystallographic characterization of Gly52Ala TmArgBP indicates that the structural context forces the residue to adopt a non-canonical conformation never observed in any of the high-medium resolution PDB structures. Interestingly, the inspection of this high resolution structure demonstrates that only minor alterations occur. Nevertheless, experiments indicate that Gly52 replacements in TmArgBP produce destabilizations comparable to those observed upon protein truncation or dissection in domains. Notably, we show that force-fields commonly used in computational biology do not reproduce this non-canonical state. Using TmArgBP as model system we here demonstrate that the structural context may force residues to adopt conformations believed to be strictly forbidden and that barely detectable alterations produce major destabilizations. Present findings highlight the role of subtle strains in governing protein stability. A full understanding of these phenomena is essential for an exhaustive comprehension of the factors regulating protein structures.

Domain communication in Thermotoga maritima Arginine Binding Protein unraveled through protein dissection

Substrate binding proteins represent a large protein family that plays fundamental roles in selective transportation of metabolites across membrane. The function of these proteins relies on the relative motions of their two domains. Insights into domain communication in this class of proteins have been here collected using Thermotoga maritima Arginine Binding Protein (TmArgBP) as model system. TmArgBP was dissected into two domains (D1 and D2) that were exhaustively characterized using a repertoire of different experimental and computational techniques. Indeed, stability, crystalline structure, ability to recognize the arginine substrate, and dynamics of the two individual domains have been here studied. Present data demonstrate that, although in the parent protein both D1 and D2 cooperate for the arginine anchoring; only D1 is intrinsically able to bind the substrate. The implications of this finding on the mechanism of arginine binding and release by TmArgBP have been discussed. Interestingly, both D1 and D2 retain the remarkable thermal/chemical stability of the parent protein. The analysis of the structural and dynamic properties of TmArgBP and of the individual domains highlights possible routes of domain communication. Finally, this study generated two interesting molecular tools, the two stable isolated domains that could be used in future investigations.

Domain swapping dissection in Thermotoga maritima arginine binding protein: How structural flexibility may compensate destabilization

Thermotoga maritima Arginine Binding Protein (TmArgBP) is a valuable candidate for arginine biosensing in diagnostics. This protein is endowed with unusual structural properties that include an extraordinary thermal/chemical stability, a domain swapped structure that undergoes large tertiary and quaternary structural transition, and the ability to form non-canonical oligomeric species. As the intrinsic stability of TmArgBP allows for extensive protein manipulations, we here dissected its structure in two parts: its main body deprived of the swapping fragment (TmArgBP20-233) and the C-terminal peptide corresponding to the helical swapping element. Both elements have been characterized independently or in combination using a repertoire of biophysical/structural techniques. Present investigations clearly indicate that TmArgBP20-233 represents a better scaffold for arginine sensing compared to the wild-type protein. Moreover, our data demonstrate that the ligand-free and the ligand-bound forms respond very differently to this helix deletion. This drastic perturbation has an important impact on the ligand-bound form of TmArgBP20-233 stability whereas it barely affects its ligand-free state. The crystallographic structures of these forms provide a rationale to this puzzling observation. Indeed, the arginine-bound state is very rigid and virtually unchanged upon protein truncation. On the other hand, the flexible ligand-free TmArgBP20-233 is able to adopt a novel state as a consequence of the helix deletion. Therefore, the flexibility of the ligand-free form endows this state with a remarkable robustness upon severe perturbations. In this scenario, TmArgBP dissection highlights an intriguing connection between destabilizing/stabilizing effects and the overall flexibility that could operate also in other proteins.

Engineering a switch-based biosensor for arginine using a Thermotoga maritima periplasmic binding protein

The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 μM across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner.

Proline 235 plays a key role in the regulation of the oligomeric states of Thermotoga maritima Arginine Binding Protein

The Arginine Binding Protein isolated from Thermotoga maritima (TmArgBP) is a protein endowed with several peculiar properties. We have previously shown that TmArgBP dimerization is a consequence of the swapping of the C-terminal helix. Here we explored the structural determinants of TmArgBP domain swapping and oligomerization. In particular, we report a mutational analysis of the residue Pro235, which is located in the hinge region of the swapping dimer. This residue was either replaced with a Gly-Lys dipeptide (TmArgBPP235GK) or a Gly residue (TmArgBPP235G).Different forms of these mutants were generated and extensively characterized using biophysical techniques.For both TmArgBPP235GK and TmArgBPP235G mutants, the occurrence of multiple oligomerization states (monomers, dimers and trimers) was detected. The formation of well-folded monomeric forms for these mutants indicates that the dimerization through C-terminal domain swapping observed in wild-type TmArgBP is driven by conformational restraints imposed by the presence of Pro235 in the hinge region. Molecular dynamics studies corroborate this observation by showing that Gly235 assumes conformational states forbidden for Pro residues in the TmArgBPP235G monomer. Unexpectedly, the trimeric forms present: (a) peculiar circular dichroism spectra, (b) a great susceptibility to heating, and (c) the ability to bind the Thioflavin T dye.The present findings clearly demonstrate that single-point mutations have an important impact on the TmArgBP oligomerization process. In a wider context, they also indicate that proteins endowed with an intrinsic propensity to swap have an easy access to states with altered structural and, possibly, functional properties.

Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins

The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity.

Studies of conformational changes of an arginine-binding protein from Thermotoga maritima in the presence and absence of ligand via molecular dynamics simulations with the coarse-grained UNRES force field.

The arginine-binding protein (ArgBP) from the hyperthermophilic eubacterium Thermotoga maritima (TmArgBP) is responsible for arginine transport through the bacterial cell membrane. The protein binds a single molecule of L-arginine, which results in conformational changes due to hinge bending. Thereby, TmArgBP acquires one of two possible conformations: open (without the presence of the arginine ligand) and closed (in the presence of the arginine ligand). Here we report a molecular dynamics study of the influence of the presence or absence of the ligand on the dynamics of TmArgBP, using the coarse-grained UNRES force field. The results of our studies indicate that binding of the arginine ligand promotes a closed conformation, which agrees with experimental data. However, the sensitivity of the TmArgBP conformation to the presence of arginine decreases and the protein becomes more flexible with increasing temperature, which might be related to the functionality of this protein in the thermophilic organism T. maritima.

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium.

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.

A loose domain swapping organization confers a remarkable stability to the dimeric structure of the arginine binding protein from Thermotoga maritima.

The arginine binding protein from Thermatoga maritima (TmArgBP), a substrate binding protein (SBP) involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate the atomic level structural properties of this intriguing protein, we determined the crystallographic structures of the apo and the arginine-bound forms of TmArgBP using MAD and SAD methods, respectively. The comparison of the liganded and unliganded models demonstrates that TmArgBP tertiary structure undergoes a very large structural re-organization upon arginine binding. This transition follows the Venus Fly-trap mechanism, although the entity of the re-organization observed in TmArgBP is larger than that observed in homologous proteins. Intriguingly, TmArgBP dimerizes through the swapping of the C-terminal helix. This dimer is stabilized exclusively by the interactions established by the swapping helix. Therefore, the TmArgBP dimer combines a high level of stability and conformational freedom. The structure of the TmArgBP dimer represents an uncommon example of large tertiary structure variations amplified at quaternary structure level by domain swapping. Although the biological relevance of the dimer needs further assessments, molecular modelling suggests that the two TmArgBP subunits may simultaneously interact with two distinct ABC transporters. Moreover, the present protein structures provide some clues about the determinants of the extraordinary stability of the biomolecule. The availability of an accurate 3D model represents a powerful tool for the design of new TmArgBP suited for biotechnological applications.

Designing a Thermostable Switch-Based Biosensor

There is a critical need for developing new diagnostic chemical reporter tools that are stable, cost effective, and capable of sensitive detection. Fluorescent protein biosensors relay changes in fluorescence based on the physical interaction between a protein and the ligand. The thermostable arginine binding protein (ArgBP) from the Gram-negative, hyperthermophilic bacterium, Thermotoga maritima, has been modified to create a reagentless fluorescent biosensor. Typically, single Cys mutants are constructed and covalently modified with environmentally-sensitive fluorescent probes. However, no theoretical framework exists to accurately model placement of the probe to the protein sensor. In this work, site-directed mutations were engineered whereby a significant fluorescence quenching (>80%) is observed based on a photo-induced electron transfer (PET) mechanism, which occurs after TmArgBP binds with increasing concentrations of arginine. This method gave significantly better fluorescence response to arginine and responded in a predictable distance-dependent manner when compared with construction of single Cys mutants of TmArgBP. Using the PET method, a dissociation constant of 0.86 microMolar was determined to be consistent with previously published data for this protein. Time-resolved fluorescence lifetime measurements indicated that fluorescence deactivation of the TmArgBP biosensor is a result of static quenching, which is consistent with the formation of ground state complexes between the fluorescent probe and an internal quencher. In addition to the development of a thermostable fluorescent protein sensor, this investigation provides a generalizable spectroscopic tool for the study of conformational movements within a single polypeptide chain.

Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein

The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic binding protein superfamily. As a highly thermostable protein, TmArgBP has been investigated for the potential to serve as a protein scaffold for the development of fluorescent protein biosensors. To establish a relationship between structural dynamics and ligand binding capabilities, we constructed single tryptophan mutants to probe the arginine binding pocket. Trp residues placed around the binding pocket reveal a strong dependence on fluorescence emission of the protein with arginine for all but one of themutants. Using these data, we calculated dissociation constants of 1.9-3.3μM for arginine. Stern-Volmer quenching analysis demonstrated that the protein undergoes a large conformational change upon ligand binding, which is a common feature of this protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic proteins and their potential biotechnological applications.

Periplasmic Binding Proteins in Thermophiles: Characterization and Potential Application of an Arginine-Binding Protein from Thermotoga maritima: A Brief Thermo-Story.

Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima with a particular eye on its potential biotechnological applications.