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In proteomics, a crucial aspect is to identify peptide sequences. De novo sequencing methods have been widely employed to identify peptide sequences, and numerous tools have been proposed over the past two decades. Recently, deep learning approaches have been introduced for de novo sequencing. Previous methods focused on encoding tandem mass spectra and predicting peptide sequences from the first amino acid onwards. However, when predicting peptides using tandem mass spectra, the peptide sequence can be predicted not only from the first amino acid but also from the last amino acid due to the coexistence of b-ion (or a- or c-ion) and y-ion (or x- or z-ion) fragments in the tandem mass spectra. Therefore, it is essential to predict peptide sequences bidirectionally. Our approach, called NovoB, utilizes a Transformer model to predict peptide sequences bidirectionally, starting with both the first and last amino acids. In comparison to Casanovo, our method achieved an improvement of the average peptide-level accuracy rate of approximately 9.8% across all species. Author summary: Understanding the characteristics of data is very important in deep learning methods. When predicting sentences, the transformer model naturally predicts from the first word. For this reason, previous methods predicted peptide sequences from the first amino acid. However, in tandem mass spectra, it is possible to predict peptide sequences bidirectionally. This method shows better results than previous approaches because it can better encode tandem mass spectra. We have demonstrated that good results can be achieved simply by understanding the characteristics of such data and using the model appropriately. We hope that this paper will help various readers improve the performance capabilities of their models. Furthermore, given that bidirectional peptide sequence prediction is crucial in de novo peptide sequence analysis, we hope that this approach will be applied to both existing and future methods utilizing deep learning techniques. [ABSTRACT FROM AUTHOR]

A protein superfold is a type of protein fold that is observed in at least three distinct, non-homologous protein families. Structural classification studies have revealed a limited number of prevalent superfolds alongside several infrequent occurring folds, and in α/β type superfolds, the C-terminal β-strand tends to favor the edge of the β-sheet, while the N-terminal β-strand is often found in the middle. The reasons behind these observations, whether they are due to evolutionary sampling bias or physical interactions, remain unclear. This article offers a physics-based explanation for these observations, specifically for pure parallel β-sheet topologies. Our investigation is grounded in several established structural rules that are based on physical interactions. We have identified "frustration-free topologies" which are topologies that can satisfy all the rules simultaneously. In contrast, topologies that cannot are termed "frustrated topologies." Our findings reveal that frustration-free topologies represent only a fraction of all theoretically possible patterns, these topologies strongly favor positioning the C-terminal β-strand at the edge of the β-sheet and the N-terminal β-strand in the middle, and there is significant overlap between frustration-free topologies and superfolds. We also used a lattice protein model to thoroughly investigate sequence-structure relationships. Our results show that frustration-free structures are highly designable, while frustrated structures are poorly designable. These findings suggest that superfolds are highly designable due to their lack of frustration, and the preference for positioning C-terminal β-strands at the edge of the β-sheet is a direct result of frustration-free topologies. These insights not only enhance our understanding of sequence-structure relationships but also have significant implications for de novo protein design. Author summary: A protein superfold is a protein fold that appears in at least three different non-homologous protein families. Superfolds are unique in their ability to accommodate multiple functions within a single fold, a feature not typically seen in other folds. Studies in structural classification have led to two notable observations: the existence of a limited number of common superfolds contrasted with a larger variety of less frequent folds, and a recurring pattern in α/β type superfolds where the C-terminal β-strand often occupies the edge of the β-sheet, while the N-terminal β-strand is usually found in the middle. The origins of these patterns, whether they stem from evolutionary sampling bias or physical interaction mechanisms, remain unclear. This article provides a physics-oriented explanation for these observations, specifically concentrating on pure parallel β-sheet topologies. The insights gained from this research are crucial in enhancing our understanding of the relationship between protein sequences and structures, and are expected to contribute significantly to the de novo design of new proteins. [ABSTRACT FROM AUTHOR]

With recent methodological advances in the field of computational protein design, in particular those based on deep learning, there is an increasing need for frameworks that allow for coherent, direct integration of different models and objective functions into the generative design process. Here we demonstrate how evolutionary multiobjective optimization techniques can be adapted to provide such an approach. With the established Non-dominated Sorting Genetic Algorithm II (NSGA-II) as the optimization framework, we use AlphaFold2 and ProteinMPNN confidence metrics to define the objective space, and a mutation operator composed of ESM-1v and ProteinMPNN to rank and then redesign the least favorable positions. Using the two-state design problem of the foldswitching protein RfaH as an in-depth case study, and PapD and calmodulin as examples of higher-dimensional design problems, we show that the evolutionary multiobjective optimization approach leads to significant reduction in the bias and variance in RfaH native sequence recovery, compared to a direct application of ProteinMPNN. We suggest that this improvement is due to three factors: (i) the use of an informative mutation operator that accelerates the sequence space exploration, (ii) the parallel, iterative design process inherent to the genetic algorithm that improves upon the ProteinMPNN autoregressive sequence decoding scheme, and (iii) the explicit approximation of the Pareto front that leads to optimal design candidates representing diverse tradeoff conditions. We anticipate this approach to be readily adaptable to different models and broadly relevant for protein design tasks with complex specifications. Author summary: Proteins are the fundamental building blocks of life, and engineering them has broad applications in medicine and biotechnology. Computational methods that seek to model and predict the protein sequence-structure-function relationship have seen significant advancement from the recent development in deep learning. As more models become available, it remains an open question how to effectively combine them into a coherent computational design approach. One approach is to perform computational design with one model, and filter the design candidates with the others. In this work, we demonstrate how an optimization algorithm inspired by evolution can be adapted to provide an alternative framework that outperforms the post hoc filtering approach, especially for problems with multiple competing design specifications. Such a framework may enable researchers to more effectively integrate the strengths of different modeling approaches to tackle more complex design problems. [ABSTRACT FROM AUTHOR]

The practical application of new single molecule protein sequencing (SMPS) technologies requires accurate estimates of their associated sequencing error rates. Here, we describe the development and application of two distinct parameter estimation methods for analyzing SMPS reads produced by fluorosequencing. A Hidden Markov Model (HMM) based approach, extends whatprot, where we previously used HMMs for SMPS peptide-read matching. This extension offers a principled approach for estimating key parameters for fluorosequencing experiments, including missed amino acid cleavages, dye loss, and peptide detachment. Specifically, we adapted the Baum-Welch algorithm, a standard technique to estimate transition probabilities for an HMM using expectation maximization, but modified here to estimate a small number of parameter values directly rather than estimating every transition probability independently. We demonstrate a high degree of accuracy on simulated data, but on experimental datasets, we observed that the model needed to be augmented with an additional error type, N-terminal blocking. This, in combination with data pre-processing, results in reasonable parameterizations of experimental datasets that agree with controlled experimental perturbations. A second independent implementation using a hybrid of DIRECT and Powell's method to reduce the root mean squared error (RMSE) between simulations and the real dataset was also developed. We compare these methods on both simulated and real data, finding that our Baum-Welch based approach outperforms DIRECT and Powell's method by most, but not all, criteria. Although some discrepancies between the results exist, we also find that both approaches provide similar error rate estimates from experimental single molecule fluorosequencing datasets. Author summary: Diverse new technologies are being developed for single-molecule protein sequencing, capable of identifying and quantifying mixtures of proteins at the level of individual molecules. There are many biochemical challenges intrinsic to high-throughput studies of proteins at such high sensitivity arising from their heterogeneous chemistries, sizes, and abundances. Beyond these challenges, the technologies themselves involve complex multi-step analytical processes. Thus, in developing and optimizing these technologies, it is important to consider the accuracy of each step and to have reliable approaches for estimating these accuracies. We focus on one particular single-molecule sequencing technology known as flourosequencing. We report and validate two methods for simultaneously determining the error-rates of each of the various steps of the fluorosequencing process. These new error estimation techniques will help researchers to better interpret the effects of changes to the chemistry and sample preparation used in fluorosequencing so that these steps can be improved. Further, more accurate determination of error rates will aid in the creation of better tools for the interpretation of this data. [ABSTRACT FROM AUTHOR]

AlphaFold2 is an Artificial Intelligence-based program developed to predict the 3D structure of proteins given only their amino acid sequence at atomic resolution. Due to the accuracy and efficiency at which AlphaFold2 can generate 3D structure predictions and its widespread adoption into various aspects of biochemical research, the technique of protein structure prediction should be considered for incorporation into the undergraduate biochemistry curriculum. A module for introducing AlphaFold2 into a senior-level biochemistry laboratory classroom was developed. The module's focus was to have students predict the structures of proteins from the MPOX 22 global outbreak virus isolate genome, which had no structures elucidated at that time. The goal of this study was to both determine the impact the module had on students and to develop a framework for introducing AlphaFold2 into the undergraduate curriculum so that instructors for biochemistry courses, regardless of their background in bioinformatics, could adapt the module into their classrooms. Author summary: AlphaFold2 is software that combines sequence similarity and structure templating with the power of Artificial Intelligence (AI) to bridge the connection between the primary protein structure (amino acid sequence) and higher-level 3D structure (secondary, tertiary, and quaternary). AlphaFold2's impressive and easily accessible nature makes it a bioinformatics tool that has been seeing a wide range of applications in biochemical research. Given this large-scale application, we examined whether Alphafold2 could be integrated into an undergraduate curriculum. We developed a novel module for a senior-level undergraduate biochemistry laboratory class. Our goal was to lay a solid foundation for other undergraduate instructors to be able to adapt this module to fit their classroom needs. While we implemented and ran all predictions on an internal university computing cluster, we recommend ColabFold for those instructors who do not have access to large-scale computational clusters or whose internal clusters cannot scale to their classroom sizes. We have outlined 3 metrics to be quantitatively investigated in the module to give both instructors and students metrics to evaluate model confidence. We have also included a template worksheet, lecture slides, and example scripts to enable instructors to rapidly develop a similar module. We hope that more departments and programs will integrate AlphaFold2 into their undergraduate curriculums, giving students a highly in-demand skill to prepare them for their transition into a career or graduate school. [ABSTRACT FROM AUTHOR]

The activities of most enzymes and drugs depend on interactions between proteins and small molecules. Accurate prediction of these interactions could greatly accelerate pharmaceutical and biotechnological research. Current machine learning models designed for this task have a limited ability to generalize beyond the proteins used for training. This limitation is likely due to a lack of information exchange between the protein and the small molecule during the generation of the required numerical representations. Here, we introduce ProSmith, a machine learning framework that employs a multimodal Transformer Network to simultaneously process protein amino acid sequences and small molecule strings in the same input. This approach facilitates the exchange of all relevant information between the two molecule types during the computation of their numerical representations, allowing the model to account for their structural and functional interactions. Our final model combines gradient boosting predictions based on the resulting multimodal Transformer Network with independent predictions based on separate deep learning representations of the proteins and small molecules. The resulting predictions outperform recently published state-of-the-art models for predicting protein-small molecule interactions across three diverse tasks: predicting kinase inhibitions; inferring potential substrates for enzymes; and predicting Michaelis constants KM. The Python code provided can be used to easily implement and improve machine learning predictions involving arbitrary protein-small molecule interactions. Author summary: Understanding how proteins interact with small molecules, such as drugs, is critical to advancing medical, biological, and biotechnological research. Our work introduces ProSmith, a machine learning framework that improves the prediction of protein-small molecule interactions. Protein-small molecule interactions can be predicted by using numerical representations of proteins and small molecules as input to machine learning prediction models. Previous methods typically generated separate numerical representations for the proteins and small molecules without considering their interactions. ProSmith, however, combines both protein sequence and small molecule structural information in the input of a single multimodal Transformer Network to generate a joint numerical representation. Unlike previous methods, this allows for a comprehensive exchange of information between protein and small molecule, capturing the complex relationships and interactions between these two types of molecules. ProSmith successfully predicts several biological interactions, including kinase inhibitions, potential enzyme-substrate pairs, and enzyme kinetic parameters KM. We provide Python code that can be easily adapted to improve predictions for any protein-small molecule interaction. [ABSTRACT FROM AUTHOR]

The design of proteins with specific tasks is a major challenge in molecular biology with important diagnostic and therapeutic applications. High-throughput screening methods have been developed to systematically evaluate protein activity, but only a small fraction of possible protein variants can be tested using these techniques. Computational models that explore the sequence space in-silico to identify the fittest molecules for a given function are needed to overcome this limitation. In this article, we propose AnnealDCA, a machine-learning framework to learn the protein fitness landscape from sequencing data derived from a broad range of experiments that use selection and sequencing to quantify protein activity. We demonstrate the effectiveness of our method by applying it to antibody Rep-Seq data of immunized mice and screening experiments, assessing the quality of the fitness landscape reconstructions. Our method can be applied to several experimental cases where a population of protein variants undergoes various rounds of selection and sequencing, without relying on the computation of variants enrichment ratios, and thus can be used even in cases of disjoint sequence samples. Author summary: Advances in sequencing techniques have recently generated an explosion of protein sequence data. This represents an opportunity for scientists to develop theoretical and computational methods that can extract relevant biological information from these data samples. In this perspective, machine learning methods are proving to be particularly effective in the biological context. Since the majority of the accessible protein sequences are not-annotated, i.e. no information about the functional properties is known, unsupervised machine learning methods are particularly suited to tackle such raw sequence data. Here, we propose an unsupervised inference method which is meant to be applied to protein sequence data generated by an evolutionary process, whether it takes place in a controlled experimental framework or in-vivo. The method is devised to be simple enough to be applied to a plethora of different experimental setups, at the same time modeling the fundamental features of the dynamical processes underlying data generation. The ultimate goal of the method is to provide a sequence-fitness mapping that goes beyond the experimentally assessed sequence space, so to assign a quantitative functional score to each possible protein variant. The accurate knowledge of this mapping is key for several biological applications, such as biomolecule design and engineering, diagnostic and therapeutic treatments, and vaccine development. [ABSTRACT FROM AUTHOR]

One of the challenges in a viral pandemic is the emergence of novel variants with different phenotypical characteristics. An ability to forecast future viral individuals at the sequence level enables advance preparation by characterizing the sequences and closing vulnerabilities in current preventative and therapeutic methods. In this article, we explore, in the context of a viral pandemic, the problem of generating complete instances of undiscovered viral protein sequences, which have a high likelihood of being discovered in the future using protein language models. Current approaches to training these models fit model parameters to a known sequence set, which does not suit pandemic forecasting as future sequences differ from known sequences in some respects. To address this, we develop a novel method, called PandoGen, to train protein language models towards the pandemic protein forecasting task. PandoGen combines techniques such as synthetic data generation, conditional sequence generation, and reward-based learning, enabling the model to forecast future sequences, with a high propensity to spread. Applying our method to modeling the SARS-CoV-2 Spike protein sequence, we find empirically that our model forecasts twice as many novel sequences with five times the case counts compared to a model that is 30× larger. Our method forecasts unseen lineages months in advance, whereas models 4× and 30× larger forecast almost no new lineages. When trained on data available up to a month before the onset of important Variants of Concern, our method consistently forecasts sequences belonging to those variants within tight sequence budgets. Author summary: Viral protein sequences play a pivotal role in the spread of a pandemic. As the virus evolves, so do the viral proteins, increasing the potency of the virus. Knowledge of future viral protein sequences can be invaluable because it allows us to test the efficacy of preventative and treatment methods against future changes to the virus, and tailor them to such changes early. We attempt to forecast viral proteins ahead of time. Making such predictions is very challenging and complex because the prediction target is a sequence with thousands of positions, and a single mis-predicted sequence position may invalidate the entire prediction. Also, as the virus continues to evolve, the data available to train models becomes obsolete. Addressing these challenges, we create a novel approach to train models of the SARS-CoV-2 Spike protein, that are especially tailored to forecasting future sequences. Models trained using this approach outperform existing approaches in their effectiveness. In addition, our method can train models to forecast important pandemic variants ahead of time. [ABSTRACT FROM AUTHOR]