Protein Language Models

Designing proteins with desired functions or properties represents a core goal in synthetic biology and drug discovery. Recent advances in protein language models (PLMs) have enabled the generation of highly designable protein sequences, while preference alignment provides a promising way to steer designs toward desired functions and properties. Nevertheless, they often trigger catastrophic forgetting of pretrained knowledge, degrading basic designability and failing to balance multiple competing objectives. To address these issues, we draw inspiration from On-Policy Distillation (OPD), an advanced post-training method renowned for mitigating catastrophic forgetting through its mode-seeking nature. In this work, we propose ProteinOPD, a multi-objective preference alignment framework that can effectively balance multiple preference objectives while maintaining the inherent designability of PLMs. ProteinOPD adapts a pretrained PLM into preference-specific teachers and distills their knowledge into a shared student via token-level OPD on the student's own trajectories. During this process, the student is aligned to a unique normalized geometric consensus of weighted teachers while ensuring bounded optimization under conflicts. This bridges the gap for OPD in multi-objective/teacher alignment. Extensive experiments show that ProteinOPD achieves substantial gains on target preference objectives without compromising the designability, with an 8x training speedup over RL-based alignment competitors.

Protein language models such as ESM-2 learn rich residue representations that achieve strong performance on protein function prediction, but their features remain difficult to interpret as structural $\&$ evolutionary signals are encoded in dense latent spaces. We propose a plug-$\&$-play framework that projects ESM-2 representations onto protein contact graphs $\&$ applies $\textbf{SoftBlobGIN}$, a lightweight Graph Isomorphism Network with differentiable Gumbel-softmax substructure pooling, to perform structure-aware message passing $\&$ learn coarse functional substructures for downstream prediction tasks. Across enzyme classification, SoftBlobGIN achieves 92.8% accuracy $\&$ 0.898 macro-F1. Unlike post hoc analysis of protein language models alone, our method produces directly auditable structural explanations: GNNExplainer recovers biologically meaningful active-site residues, spatially localized functional clusters, $\&$ catalytic contact patterns. On binding-site detection, SoftBlobGIN improves residue AUROC from $0.885$ using an ESM-2 linear probe to $0.983$, indicating that these structural explanations are not recoverable from language-model features alone. Learned blob partitions provide an additional layer of interpretability by automatically grouping residues into functional substructures, with blobs containing annotated active-site residues showing $1.85\times$ higher importance than other blobs ($ρ{=}0.339$, $p{=}0.009$), without any active-site supervision. Our framework requires no retraining of the language model, adds only $\sim$1.1M parameters, $\&$ generalises across ProteinShake tasks, achieving $F_{\max}$ of $0.733$ on Gene Ontology prediction $\&$ AUROC of $0.969$ on binding-site detection. We position this as an interpretable structural companion to protein language models that makes their predictions more transparent $\&$ auditable.

Protein language models are trained primarily with masked language modeling (MLM), which predicts masked amino-acid identities. Joint-embedding predictive architectures (JEPA) instead predict latent representations, but have not been applied to proteins. ProteinJEPA supplements MLM with a cosine loss for predicting the half-depth hidden states of a teacher given the unmasked sequence. On 19 tasks, with ESM2 at 35M and 150M parameters and three pretraining seeds, MLM+JEPA outperforms compute-matched and step-matched MLM-only continued training in 78 and 76 of 114 comparisons (14 losses, 22 ties). The median compute-matched gain is $+0.0106$ on structure- and homology-sensitive tasks versus $+0.0041$ elsewhere, led by SCOPe-40 retrieval and remote homology with improvements of 6.1 percentage points in Recall@1 and 2.7 points in accuracy, respectively. Gains on these tasks increase with model size from 8M to 150M. Against the off-the-shelf checkpoint, MLM+JEPA wins 81 of 114 comparisons (median $+0.0068$) without improving MLM loss. In random initialization the gain is smaller and replicates inconsistently across seeds ($p{=}0.059$). The same recipe improves the causal ProGen3 model, beating a compute-matched next-token-prediction control on 12 of 16 tasks. Ablations show that cosine loss beats mean squared error, while adding shallower targets removes most of the task gain. JEPA-only training collapses downstream performance: latent prediction complements MLM rather than replacing it. Code: https://anonymous.4open.science/r/protJepa-FF24

Protein language models (pLMs) produce per-residue representations that capture evolutionary and structural information, yet their mean-pooled sequence embeddings are not explicitly trained to reflect functional, evolutionary or structural similarity between proteins. We present Protein Sentence Transformers (ProtSent), a contrastive fine-tuning framework for adapting PLMs into general-purpose embedding models. ProtSent trains with MultipleNegativesRankingLoss across five protein-pair datasets: Pfam families, structurally derived hard negatives, AlphaFold DB structural pairs, and StringDB protein--protein interactions, and Deep Mutational Scanning data. We evaluate on 23~downstream tasks using frozen embeddings with a k-nearest-neighbor probe to measure embedding neighborhood quality. On ESM-2 150M, ProtSent improves 15 of 23 tasks, with gains of +105% on remote homology detection, +17% on variant effect prediction, and +19.9% Recall@1 on SCOPe-40 structural retrieval. The 35M variant improves 16 of 23 tasks with +40.5% on remote homology and +15.5% Recall@1 on SCOPe-40. Contrastive fine-tuning restructures the embedding space to better capture protein function and structure, without any task-specific supervision. We release the models, public data, and training recipe and code.

Antibody therapeutics are among the most successful modern medicines, yet computationally designing antibodies with desirable binding and developability properties remains challenging. While protein language models (pLMs) have emerged as powerful tools for antibody sequence design, existing approaches largely suffer from two key limitations: they predominantly memorize germline sequences rather than modeling biologically meaningful somatic variation, and they offer limited support for flexible classifier-guided conditional generation. We address these challenges through two primary contributions. First, we demonstrate that discrete diffusion fine-tuning achieves strong language modeling performance on antibody sequences while allowing for generation conditioned on any off-the-shelf classifier. Second, we introduce germline absorbing diffusion, a novel modification of the discrete diffusion noise process in which the germline sequence - rather than a masked sequence - serves as the absorbing state. This biologically motivated inductive bias restricts the model to learning the trajectory from germline to observed sequence, effectively excluding genetic variation and V(D)J recombination statistics from the learned distribution and dramatically mitigating germline bias. We show that germline diffusion improves non-germline residue prediction accuracy from 26 percent to 46 percent, approaching the theoretical upper bound set by true biological variability. We then demonstrate the utility of our germline diffusion model on the conditional generation tasks of sampling antibodies with improved hydrophobicity and predicted binding affinity. On both tasks our model shows an improved tradeoff between class adherence and sample quality, significantly outperforming EvoProtGrad, a popular strategy to sample from pLMs with gradient-based discrete Markov Chain Monte Carlo.

Protein language models are increasingly used to guide experimental and clinical decisions, yet it is often unclear whether a confident prediction reflects recognition of biological evidence or retrieval of a statistical default. We examine this distinction for a near-universal biological rule, that proteins begin with methionine, by tracing the computational pathway through which ESM2-8M produces this prediction. The model does not detect methionine at the masked position. Instead, it retrieves a methionine-favouring signal from a reference representation at the beginning-of-sequence token via a position-specific query assembled across layers, with the final output emerging through competition with context-dependent circuits. To understand how positional information reaches the readout, we introduce a norm-direction decomposition of attention scores within rotary frequency bands. Positional encoding operates through coupled changes in query norm and angular alignment distributed across these bands. On sequences whose true N-terminus is not methionine, where the biological question matters, the model predicts methionine anyway. This is not a correct prediction produced by an unexpected mechanism, but the output of a positional-prior retrieval circuit that matches the statistical average and fails where biology diverges from it. Distinguishing the two requires resolution at the level of individual circuits, frequency bands, and query composition, suggesting that mechanistic verification will be necessary, and challenging, for predictions where the biological stakes are higher. Even for the simplest biological rule, the model's prediction is mediated by a distributed computational circuit rather than direct recognition, suggesting that increasing task complexity will further obscure the relationship between model confidence and underlying biological evidence.

De novo functional protein design aims to generate protein sequences that realize specified biochemical functions without relying on evolutionary templates, enabling broad applications in biotechnology and medicine. Existing approaches adopt either direct function-to-sequence mapping or decoupled structure-sequence generation strategies but often fail to achieve functionality and foldability simultaneously. To address this, we propose CodeFP, a Co-generative protein language model for de novo Functional Protein design that simultaneously decodes sequence and structure tokens, thereby enabling superior simultaneous realization of functionality and foldability. CodeFP utilizes functional local structures to enrich functional semantic encodings, overcoming the suboptimal translation of flat encodings into structure tokens, while introducing auxiliary functional supervision to alleviate training ambiguity stemming from the one-to-many structure-to-token mapping. Extensive experiments show that CodeFP consistently achieves average improvements of 6.1% in functional consistency and 3.2% in foldability over the strongest baseline.

Proteins are shaped by gradual evolution under biophysical and functional constraints. Protein language models learn rich evolutionary constraints from large-scale sequences, and discrete diffusion-based protein language models~(\eg, DPLMs) are promising for both understanding and generation. However, existing DPLMs typically rely on masked diffusion that contradicts a simple biological intuition: proteins evolve through accumulated edits, not by emerging from masks. Consequently, these frameworks lack explicit pretraining objectives for substitution and insertion/deletion (indel) operations, limiting both optimization-style post-editing and flexible guided generation. To address these limitations, we present DPLM-Evo, an evolutionary discrete diffusion framework that explicitly predicts substitution, insertion, and deletion operations during denoising. DPLM-Evo decouples an upsampled-length latent alignment space from the variable-length observed sequence space, which makes indel-aware generation tractable. To better align substitutions with real evolution, we further introduce a contextualized evolutionary noising kernel that produces biologically informed, context-dependent mutation patterns. Across tasks, DPLM-Evo improves sequence understanding and achieves state-of-the-art mutation effect prediction performance on ProteinGym in the single-sequence setting. It also enables variable-length simulated evolution, and post-editing/optimization of existing proteins via explicit edit trajectories.

Using a statistical mechanics framework, we investigate the parameter space of transformer models trained on protein sequence data. We sample the loss landscape at varying temperatures using Langevin dynamics to characterize the low-loss manifold, and to understand the mechanisms underlying transformers' superior performance in protein structure prediction. We find that, at variance with networks not based on the attention mechanism, the lack of a first--order--like transition in the loss of the transformer produces a range of intermediate temperatures with good learning properties; this is true both for synthetic and natural protein sequences. We also show that the parameters of most layers are highly conserved at these temperatures if the dimension of the embedding is optimal, and we provide an operative way to find this dimension. Additionally, we show that the attention matrix is more predictive of the contact maps of the protein at higher temperatures and for higher dimensions of the embedding than those optimal for learning. Finally, we showed that the models sampled at intermediate temperatures can predict the free-energy variation upon mutation, better than models obtained through standard optimization techniques.

Protein generative models have shown remarkable promise in protein design, yet their success rates remain constrained by reliance on curated sequence-structure datasets and by misalignment between supervised objectives and real design goals. We present ProteinZero, an online reinforcement learning framework for inverse folding models that enables scalable, automated, and continuous self-improvement with computationally efficient feedback. ProteinZero employs a reward pipeline that combines structural guidance from ESMFold with a novel self-derived ddG predictor, providing stable multi-objective signals while avoiding the prohibitive cost of physics-based methods. To ensure robustness in online RL, we further introduce a novel embedding-level diversity regularizer that mitigates mode collapse and promotes sequence-level diversity among generated designs. Within a general RL formulation balancing multi-reward optimization, KL-divergence from a reference model, and diversity regularization, ProteinZero achieves robust improvements across designability, predicted stability, recovery, and diversity. On the CATH-4.3 benchmark, it consistently outperforms state-of-the-art baselines including ProteinMPNN, ESM-IF, and InstructPLM, reducing design failure rates by 36-48% and achieving success rates above 90% across diverse folds. Importantly, a complete RL run can be executed on a single 8xGPU node within three days, including reward computation and data generation. These results indicate that efficient online RL fine-tuning can complement supervised pretraining by allowing protein generative models to evolve continuously from their own outputs and optimize multiple design objectives without labeled data, opening new possibilities for exploring the vast protein design space. Code and model checkpoints are available at https://github.com/ziwenwang28/ProteinZero.

Natural Language Processing (NLP) has transformed various fields beyond linguistics by applying techniques originally developed for human language to the analysis of biological sequences. This review explores the application of NLP methods to biological sequence data, focusing on genomics, transcriptomics, and proteomics. We examine how various NLP methods, from classic approaches like word2vec to advanced models employing transformers and hyena operators, are being adapted to analyze DNA, RNA, protein sequences, and entire genomes. The review also examines tokenization strategies and model architectures, evaluating their strengths, limitations, and suitability for different biological tasks. We further cover recent advances in NLP applications for biological data, such as structure prediction, gene expression, and evolutionary analysis, highlighting the potential of these methods for extracting meaningful insights from large-scale genomic data. As language models continue to advance, their integration into bioinformatics holds immense promise for advancing our understanding of biological processes in all domains of life.

Modeling protein sequences as a language has made language models a powerful tool in computational biology, yet the language itself remains poorly understood. A key step toward understanding it is identifying its constituent units. In natural languages, morphemes can occur in multiple forms; similarly, in proteins, mutations can give rise to variations of a unit that persist through evolution, forming families of related units. We introduce PUMA (Protein Units via Mutation-Aware Merging), an algorithm that learns protein units from sequence and explores their mutational variants using substitution matrices, forming a genealogy of unit families. Our results show that mutations remaining within a PUMA family are more often benign than the substitution matrix alone predicts, and that PUMA genealogy improves molecular function representations compared to treating units independently. A case study of a unit family demonstrates relatedness beyond homology. PUMA achieves competitive performance on downstream tasks when used as a protein language model tokenizer. Moreover, collapsing units into families results in a smaller embedding table and faster training. Together, these results support PUMA as a biologically grounded protein vocabulary that organizes protein units into plausible families of mutational variants. The source code is available at https://github.com/boun-tabi-lifelu/PUMA.

\textbf{Motivation:} Pre-trained protein language models (PLMs) such as ESM-2 have become the default representation for biological sequence tasks, but they are computationally heavy and require GPUs both for embedding and for fine-tuning. Whether they are actually necessary for sequence \emph{classification}, as opposed to structure prediction, is rarely tested against strong, principled, lightweight alternatives. This question has direct practical stakes for large-scale genomic surveillance, where embedding millions of sequences on commodity hardware is a recurring bottleneck.\ \textbf{Results:} We introduce Murmur2Vec, an alignment-free, training-free embedding that aggregates $k$-mer counts into a small hash table via the deterministic MurmurHash function, and we cast it as a randomized sketch of the classical $k$-mer spectrum kernel. We provide a complete theoretical treatment: closed-form bias/variance of the inner product, an unbiased signed variant with a Johnson--Lindenstrauss-type concentration bound, an excess-risk bound for downstream linear classifiers that makes the bias--variance trade-off in the hash-table size explicit, and an implicit-regularization mechanism by which collisions damage frequent non-discriminative $k$-mers more than rare lineage-defining ones. Across four classification tasks, SARS-CoV-2 spike lineage (22 classes), HIV-1 Env subtype (8 classes), and two protein-family benchmarks (8 and 6 classes), Murmur2Vec matches a LoRA-fine-tuned 650M-parameter ESM-2 model on the two tasks for which LoRA fine-tuning was run to convergence (SARS-CoV-2 and HIV-1) and ties frozen ESM-2 on the two protein-family tasks, and it \emph{outperforms} the fine-tuned model on the hardest task (SARS-CoV-2 lineage: $0.854$ vs.\ $0.807$ accuracy; macro-F1 $0.684$ vs.\ $0.401$).