Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding and function.

Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.

Voltage vs. Ligand I: Structural basis of the intrinsic flexibility of S3 segment and its significance in ion channel activation.

We systematically predict the internal flexibility of the S3 segment, one of the most mobile elements in the voltage-sensor domain. By analyzing the primary amino acid sequences of V-sensor containing proteins, including Hv1, TPC channels and the voltage-sensing phosphatases, we established correlations between the local flexibility and modes of activation for different members of the VGIC superfamily. Taking advantage of the structural information available, we also assessed structural aspects to understand the role played by the flexibility of S3 during the gating of the pore. We found that S3 flexibility is mainly determined by two specific regions: (1) a short NxxD motif in the N-half portion of the helix (S3a), and (2) a short sequence at the beginning of the so-called paddle motif where the segment has a kink that, in some cases, divide S3 into two distinct helices (S3a and S3b). A good correlation between the flexibility of S3 and the reported sensitivity to temperature and mechanical stretch was found. Thus, if the channel exhibits high sensitivity to heat or membrane stretch, local S3 flexibility is low. On the other hand, high flexibility of S3 is preferentially associated to channels showing poor heat and mechanical sensitivities. In contrast, we did not find any apparent correlation between S3 flexibility and voltage or ligand dependence. Overall, our results provide valuable insights into the dynamics of channel-gating and its modulation.

Structure and conformational stability of the triosephosphate isomerase from Zea mays. Comparison with the chemical unfolding pathways of other eukaryotic TIMs.

We studied the structure, function and thermodynamic properties for the unfolding of the Triosephosphate isomerase (TIM) from Zea mays (ZmTIM). ZmTIM shows a catalytic efficiency close to the diffusion limit. Native ZmTIM is a dimer that dissociates upon dilution into inactive and unfolded monomers. Its thermal unfolding is irreversible with a Tm of 61.6 ±â€¯1.4 °C and an activation energy of 383.4 ±â€¯11.5 kJ mol-1. The urea-induced unfolding of ZmTIM is reversible. Transitions followed by catalytic activity and spectroscopic properties are monophasic and superimposable, indicating that ZmTIM unfolds/refolds in a two-state behavior with an unfolding ΔG°(H20) = 99.8 ±â€¯5.3 kJ mol-1. This contrasts with most other studied TIMs, where folding intermediates are common. The three-dimensional structure of ZmTIM was solved at 1.8 Å. A structural comparison with other eukaryotic TIMs shows a similar number of intramolecular and intermolecular interactions. Interestingly the number of interfacial water molecules found in ZmTIM is lower than those observed in most TIMs that show folding intermediates. Although with the available data, there is no clear correlation between structural properties and the number of equilibrium intermediates in the unfolding of TIM, the identification of such structural properties should increase our understanding of folding mechanisms.

Side chain flexibility and coupling between the S4-S5 linker and the TRP domain in thermo-sensitive TRP channels: Insights from protein modeling.

The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side-chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4-S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly-conserved Trp during this coupling. Our analysis suggests an important role of the S4-S5L flexibility in the thermosensitivity, where heat-activated channels possess rigid S4-S5 linkers, whereas cold-activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4-S5L, importantly the central Pro. Proteins 2017; 85:630-646. © 2016 Wiley Periodicals, Inc.

Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (ß/α)8 barrel subunits, albeit this "TIM barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery.