Better Protein Function Prediction by Modeling Survivorship Bias
Authors: Zhongmou Chao, Poompol Buathong, Ekaterina Selivanovitch, Susan Daniel, Peter I. Frazier
Organizations: Smith School of Chemical and Biomolecular Engineering, Cornell University, USA · Center for Applied Mathematics, Cornell University, USA · School of Operations Research and Information Engineering, Cornell University, USA
Protein sequence data from nature exhibits survivorship bias: we only observe data from those organisms that survive and reproduce, while non-functional protein mutations are eliminated by natural selection. Thus, predicting whether a protein sequence is functional often requires learning from positive examples alone. While positive-unlabeled (PU) learning frameworks offer a generic solution to this problem, existing PU methods ignore the evolutionary processes that shape sequence observability and cause survivorship bias. Consider a sequence that is one mutation away from a commonly-observed protein variant in a well-surveilled organism. If the sequence were functional, it would likely be observed. If it is not observed, this suggests non-functionality. In contrast, sequences that are unlikely to arise through mutation may be missing simply because they never arose. Thus, these two kinds of missing sequences should be treated differently when training models. In this work, we propose Evo-PU, a PU learning framework that uses a scientific understanding of nucleotide mutation to model survivorship bias for well-surveilled single-organism sequence data. On three prediction tasks using single-organism uniform-coverage surveillance data -- predicting results from held-out influenza and respiratory syncytial virus (RSV) mutagenesis studies, and predicting future SARS-CoV-2 variants -- Evo-PU outperforms standard PU learning, one-class classification (OCC), and protein language models (PLMs). On prediction tasks from multi-organism ProteinGym datasets with more heterogeneous surveillance coverage, we identify opportunities to generalize our approach.
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.
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.
Variational autoencoders (VAEs) trained on multiple sequence alignments (MSAs) have emerged as powerful generative models for biological sequences, with applications ranging from disease variant prediction to functional RNA design. However, standard biological VAE training treats all sequences as exchangeable, ignoring the rich evolutionary structure that organizes homologous sequences from evolutionarily close to highly divergent. We propose Evolutionary Curriculum Learning (ECL), a training strategy that exploits this structure by progressively exposing the model to sequences of increasing evolutionary distance from sampled anchors, following a power-law expansion schedule. Applied to two architecturally distinct VAE models and two biological domains--protein variant effect prediction with EVE and RNA family sequence generation with RfamGen--ECL improves downstream task performance across five random seeds per configuration. Mean ClinVar classification AUROC rises from 0.981 to 0.989 for p53; for PTEN, ECL attains 1.000 in every seed whereas the baseline is unstable (mean 0.905, falling as low as 0.54). For RNA, ECL raises mean covariance-model bit scores on all three families tested and exceeds its seed-matched baseline in 12 of 15 training runs, though with only three families the effect cannot be established as significant at the family level. Ablation experiments show that progressively expanding the sampled sequences by evolutionary distance outperforms fixed-size neighborhood sampling in addition to uniform random sampling. Evolutionary distance is therefore a useful inductive bias for ordering the training curriculum in biological sequence modeling.