Stepwise introduction of stabilizing mutations reveals nonlinear additive effects in de novo TIM barrels.

Over the past decades, the TIM-barrel fold has served as a model system for the exploration of how changes in protein sequences affect their structural, stability, and functional characteristics, and moreover, how this information can be leveraged to design proteins from the ground up. After numerous attempts to design de novo proteins with this specific fold, sTIM11 was the first validated de novo design of an idealized four-fold symmetric TIM barrel. Subsequent efforts to enhance the stability of this initial design resulted in the development of DeNovoTIMs, a family of de novo TIM barrels with various stabilizing mutations.

Exploring the Protein Sequence Space with Global Generative Models.

Recent advancements in specialized large-scale architectures for training images and language have profoundly impacted the field of computer vision and natural language processing (NLP). Language models, such as the recent ChatGPT and GPT-4, have demonstrated exceptional capabilities in processing, translating, and generating human language. These breakthroughs have also been reflected in protein research, leading to the rapid development of numerous new methods in a short time, with unprecedented performance.

Retracing the evolution of a modern periplasmic binding protein.

Investigating the evolution of structural features in modern multidomain proteins helps to understand their immense diversity and functional versatility. The class of periplasmic binding proteins (PBPs) offers an opportunity to interrogate one of the main processes driving diversification: the duplication and fusion of protein sequences to generate new architectures. The symmetry of their two-lobed topology, their mechanism of binding, and the organization of their operon structure led to the hypothesis that PBPs arose through a duplication and fusion event of a single common ancestor.

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.

Structures of permuted halves of a modern ribose-binding protein.

Periplasmic binding proteins (PBPs) are a class of proteins that participate in the cellular transport of various ligands. They have been used as model systems to study mechanisms in protein evolution, such as duplication, recombination and domain swapping. It has been suggested that PBPs evolved from precursors half their size.

A newly introduced salt bridge cluster improves structural and biophysical properties of de novo TIM barrels.

Protein stability can be fine-tuned by modifying different structural features such as hydrogen-bond networks, salt bridges, hydrophobic cores, or disulfide bridges. Among these, stabilization by salt bridges is a major challenge in protein design and engineering since their stabilizing effects show a high dependence on the structural environment in the protein, and therefore are difficult to predict and model. In this work, we explore the effects on structure and stability of an introduced salt bridge cluster in the context of three different de novo TIM barrels.

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (β/⍺)-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis.

Evolution, folding, and design of TIM barrels and related proteins.

Proteins are chief actors in life that perform a myriad of exquisite functions. This diversity has been enabled through the evolution and diversification of protein folds. Analysis of sequences and structures strongly suggest that numerous protein pieces have been reused as building blocks and propagated to many modern folds.

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.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels.

In the preceding article, we present a flexibility analysis of the voltage-gated ion channel (VGIC) superfamily. In this study, we describe in detail the flexibility profile of the voltage-sensor domain (VSD) and the pore domain (PD) concerning the evolution of 6TM ion channels. In particular, we highlight the role of flexibility in the emergence of CNG channels and describe a significant level of sequence similarity between the archetypical VSD and the TolQ proteins.

FRET-based analysis and molecular modeling of the human GPN-loop GTPases 1 and 3 heterodimer unveils a dominant-negative protein complex.

GPN-loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1-Gpn3 interaction in live HEK293-AD cells.

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox-based modifications.

Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation.

A strategy based on thermal flexibility to design triosephosphate isomerase proteins with increased or decreased kinetic stability.

Kinetic stability of proteins determines their susceptibility to irreversibly unfold in a time-dependent process, and therefore its half-life. A residue displacement analysis of temperature-induced unfolding molecular dynamics simulations was recently employed to define the thermal flexibility of proteins. This property was found to be correlated with the activation energy barrier (E) separating the native from the transition state in the denaturation process.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.

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.