Biology subjects

Romero-Romero, S.

Publications and source records attributed to Romero-Romero, S..

6 recordsLinked to original sources

bioRxiv2024

De novo design of triosephosphate isomerases using generative language models

The design of proteins with tailored functions is of immense interest to biotechnology, medicine, and the chemical industry. While protein design is rapidly evolving with the use of AI techniques, the design of complex enzymes remains a challenge. Here, we present the use of two large language models (LLMs), ZymCTRL and ProtGPT2, for the generation of de novo enzymes that catalyze the triosephosphate isomerase (TIM) reaction. Natural TIM enzymes are obligatory oligomers that catalyze a multi-step isomerization reaction near the diffusion limit. This makes TIM an ideal target to assess the generative ability of protein language models. Newly generated sequences were filtered to obtain a set of twelve candidates from each approach for experimental validation. Multiple constructs from both language models exhibit the intended function in vivo through their ability to complement a TIM-deficient E. coli strain. In-depth characterization of the best-behaving artificial enzyme reveals behavior and catalytic efficiency close to its natural counterparts. These findings support the use of conditional and fine-tuned unconditional LLMs for the generation of complex enzymes.

biochemistry↗

bioRxiv2024

Code for Collagen Folding Deciphered

Collagen triple helix folds in two steps: nucleation of three polypeptides at the C-termini followed by zip-chain like propagation. The triple helices found in all domains of life as well as viruses contain upto 6000 amino acids in each polypeptide that are also frequently interrupted with non-helical sequences that disrupt folding and reduce stability. Given the length of polypeptide and the disruptive interruptions, compensating mechanisms that stabilize against local unfolding during propagation and offset the entropic cost of folding the long polypeptides are not fully understood. Here, we show that the information for correct folding of collagen triple helices is encoded in their sequence as interchain electrostatic interactions. In case of humans, disrupting these interactions causes severe to lethal diseases. Key ResultCollagen triple helices found in all the three domains of life as well as viruses have converged on similar mechanism to fold correctly.

biochemistry↗

bioRxiv2023

Hydrophobic Clusters Direct Folding of a Synthetic Chimeric Protein

Extant proteins frequently share sub-domain sized fragments, suggesting that among other mechanisms, proteins evolved new structure and functions via recombination of existing fragments. While the role of protein fragments as evolutionary units is well-established, their biophysical features necessary for generating a well-folded and stable protein are not clearly understood. In order to probe how fragments determine foldability and stability of recombined proteins, we investigated the stability, folding and dynamics of a synthetic chimera created by fusion of fragments of the chemotactic response regulator protein CheY that belongs to the flavodoxin-like fold and imidazole glycerol phosphate synthase from histidine biosynthesis (HisF) which harbors the TIM-barrel fold. The chimera unfolds via an equilibrium intermediate. Mutation of a glycine residue present at the interface of the CheY and HisF fragments to a valine abrogates the equilibrium intermediate while mutation to isoleucine dramatically increases the native state kinetic stability without any significant change in the folding rate. Parts of the fragment interface in the chimera are found to be conformationally dynamic while hydrophobic mutations globally increase its conformational rigidity. We hypothesize that the hydrophobic mutation improves sidechain packing in a large cluster of isoleucine, leucine and valine (ILV) residues that spans the fragment interface. We also extrapolate that inheritance of large ILV clusters from parent proteins could be a key determinant of successful fragment recombination.

biophysics↗

bioRxiv2023

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. To investigate this claim, we set out to reverse the evolutionary process and recreate the structural equivalent of a single-lobed progenitor using ribose-binding protein (RBP) as our model. We found that this modern PBP can be deconstructed into its lobes, producing two proteins that represent possible progenitor halves. The isolated halves of RBP are well folded and monomeric proteins, albeit with a lower thermostability, and do not retain the original binding function. However, the two entities readily form a heterodimer in vitro and in-cell. The X-ray structure of the heterodimer closely resembles the parental protein. Moreover, the binding function is fully regained upon formation of the heterodimer with a ligand affinity similar to that observed in the modern RBP. This highlights how a duplication event could have given rise to a stable and functional PBP-like fold and provides insights into how more complex functional structures can evolve from simpler molecular components.

biochemistry↗

bioRxiv2023

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

Substrate-binding proteins (SBP) are used by organisms from the three domains of life for transport and signaling. 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 binding protein (LAO) 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 behavior 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.

biochemistry↗

bioRxiv2020

Epistasis on 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. High conformational stability lowers protein sensitivity to mutations and changes in the environment; thus, understanding and manipulating protein stability will expand the applications of de novo proteins. Since the ({beta}/)8-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. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design. Significance StatementDe novo protein design expands our knowledge about protein structure and stability. The TIM barrel is a highly relevant fold used in nature to host a rich variety of catalytic functions. Here, we follow a modular approach to design and characterize a collection of de novo TIM barrels and subjected them to a thorough folding analysis. Non-additive effects modulate the increase in stability when different regions of the barrel are mutated, showing a wide variety of thermodynamic properties that allow them to navigate an unexplored region of the stability landscape found in natural TIM barrels. The design of stable proteins increases the applicability of de novo proteins and provides crucial information on the molecular determinants that modulate structure and stability. One Sentence SummaryA family of designed TIM barrels with diverse thermodynamic properties shows epistatic effects on its stability landscape.

biochemistry↗