DOI: 10.5061/dryad.5dv41nsjg
title: Structural models and Sort-seq data for: Packing of apolar amino acids is not a strong stabilizing force in transmembrane helix dimerization

description: The factors that stabilize the folding and oligomerization of membrane
 proteins are still not well understood. In particular, it remains unclear
 how the tight and complementary packing between apolar side chains
 observed in the core of membrane proteins contributes to their stability.
 Complementary packing is a necessary feature since packing defects are
 generally destabilizing for membrane proteins. The question is the extent
 to which packing of apolar side chains – and the resulting van der Waals
 interactions – is a sufficient driving force for stabilizing the
 interaction between transmembrane helices in the absence of hydrogen
 bonding and polar interactions. We addressed this question with an
 approach based on high-throughput protein design and the homodimerization
 of single-pass helices as the model system. We designed hundreds of
 transmembrane helix dimers mediated by apolar packing in the backbone
 configurations that are most commonly found in membrane proteins. We
 assessed the association propensity of the designs in the membrane of
 Escherichia coli and found that they were most often monomeric or, at
 best, weakly dimeric. Conversely, a set of controls designed in the
 backbone configuration of the GASright motif, which is mediated by weak
 hydrogen bonds, displayed significantly higher dimerization propensity.
 The data suggest that packing of apolar side chains and van der Waals
 interactions are a relatively weak force in driving transmembrane helix
 dimerization. It also confirms that GASright is a special configuration
 for achieving stability in membrane proteins.
description: # Structural models and Sort-seq data for: Packing of apolar amino acids
 is not a strong stabilizing force in transmembrane helix dimerization This
 repository contains data relative to Loiseau and Senes, bioRxiv article
 [https://doi.org/10.1101/2025.04.26.649789](https://doi.org/10.1101/2025.04.26.649789) * Structural model of designed dimers (PDB files) * Sort-seq Data --- ## Structural model of dimers (PDB files) The compressed zip file contains the structural models of the designed transmembrane dimers. The file name corresponds to the constructs listed in Table S1: [G/R/L]_NNN.pdb, where * G identifies the GAS-right dimers * L the Left dimers * R the Right dimers NNN is an integer serial number. ### File * `structural_models.zip` ### Code/software PDB files are viewable with PyMol or other software that can read Protein Data Bank coordinate files. --- ## NGS Data Raw data from Next Generation Sequencing utilizing the TOXGREEN sort-seq methods as in Anderson et al., 2025 doi: [https://doi.org/10.1101/2025.04.22.650048](https://doi.org/10.1101/2025.04.22.650048). ### Oligo pool segment data ##### File refSeqs.csv This CSV file contains the segment in the oligo pool library, the protein sequence, and the DNA sequence of all constructs. These include the designs (supplementary Table S3), their mutants, and the control sequences used to calibrate the TOXGREEN sort-seq runs. | Column Name | Content | | ---------------- | ------------------------------------------------------------------------------------------------------------------------------------ | | Segment number | Number of the segment in which the protein was coded (C=calibration control, P=Positive Maltose Control, N=Negative Maltose Control) | | Protein sequence | Protein sequence | | DNA sequence | DNA sequence ordered | #### Raw TOXGREEN Sort-seq NGS Data Raw NGS sequencing counts are provided in the following files: * `Counts_sort.csv` * `Percentage_sort.csv` These data were used to reconstruct the fluorescence profiles for all design and mutant sequences ("sort” files, Fig. 2C-D and Fig. 3). ##### File Count_sort.csv Counts of constructs sorted in NGS | Column Name | Content | | ----------- | ------------------------------------------------------------------------------------------------------------------------------------ | | Sequence | Protein Sequence | | Segment | Number of the segment in which the protein was coded (C=Calibration control, P=Positive Maltose Control, N=Negative Maltose Control) | | G1-Rep1 | Counts for GASright constructs sorted into bin 1, replicate 1 | | G1-Rep2 | Counts for GASright constructs sorted into bin 1, replicate 2 | | … | 
 
 | | L1-Rep1 | Counts for Left constructs sorted into bin 1, replicate 1 |
 | L2-Rep2 | Counts for Left constructs sorted into bin 1, replicate 2 | |
 … | 
 
 | | R1-Rep1 | Counts for Right constructs sorted into bin 1, replicate 1
 | | R2-Rep2 | Counts for Right constructs sorted into bin 1, replicate 2 |
 | … | 
 
 | ##### File Percentage_sort.csv Percent total reads of constructs sorted
 in NGS | Column Name | Content | | ----------- |
 ------------------------------------------------------------------------------------------------------------------------------------ | | Sequence | Protein Sequence | | Segment | Number of the segment in which the protein was coded (C=Calibration control, P=Positive Maltose Control, N=Negative Maltose Control) | | G1-Rep1 | Percent total reads for GASright constructs sorted into bin 1, replicate 1 | | G1-Rep2 | Percent total reads for GASright constructs sorted into bin 1, replicate 2 | | … | 
 
 | | L1-Rep1 | Percent total reads for Left constructs sorted into bin 1,
 replicate 1 | | L2-Rep2 | Percent total reads for Left constructs sorted
 into bin 1, replicate 2 | | … | 
 
 | | R1-Rep1 | Percent total reads for Right constructs sorted into bin 1,
 replicate 1 | | R2-Rep2 | Percent total reads for Right constructs sorted
 into bin 1, replicate 2 | | … | 
 
 | ###### *Naming scheme:* G# = Sequence counts for GASright constructs in
 a sort-seq bin*\ L# = Sequence counts for Left constructs in a sort-seq
 bin*\ R# = Sequence counts for Right constructs in a sort-seq bin*\ Rep# =
 Replicate number\ *Bin numbers in ascending order correspond to increasing
 fluorescence intensity ## NSG data from MalE Complementation Assay Raw
 data from Next Generation Sequencing for the MalE Complementation assay,
 utilizing the TOXGREEN sort-seq methods as in Anderson et al., 2025 doi:
 [https://doi.org/10.1101/2025.04.22.650048](https://doi.org/10.1101/2025.04.22.650048). The corresponding relative frequencies of the above counts for each construct are provided in the following files: * `Count_selection.csv` * `Percentage_selection.csv` These data were used to select the final set of design constructs of design ("sort” files, Fig. 2C-D and Fig. 3). ##### File Count_selection.csv Counts of constructs from the Maltose test, used for selection | Column Name | Content | | ----------- | ------------------------------------------------------------------------------------------------------------------------------------ | | Sequence | Protein Sequence | | Segment | Number of the segment in which the protein was coded (C=Calibration control, P=Positive Maltose Control, N=Negative Maltose Control) | | G1-LB-0H | Counts for GASright constructs grown in LB media, replicate 1 at 0 hours | | G1-LB-12H | Counts for GASright constructs grown in LB media, replicate 1 at 12 hours | | … | 
 
 | | G1-M9-30H | Counts for GASright constructs grown in Maltose media,
 replicate 1 at 30 hours | | … | 
 
 | | L1-LB-0H | Counts for Left constructs grown in LB media, replicate 1
 at 0 hours | | R1-LB-0H | Counts for Right constructs grown in LB media,
 replicate 1 at 0 hours | ##### File Percentage_selection.csv Percent total
 reads of constructs from the Maltose test, used for selection | Column
 Name | Content | | ----------- |
 ------------------------------------------------------------------------------------------------------------------------------------ | | Sequence | Protein Sequence | | Segment | Number of the segment in which the protein was coded (C=Calibration control, P=Positive Maltose Control, N=Negative Maltose Control) | | G1-LB-0H | Percent total reads for GASright constructs grown in LB media, replicate 1 at 0 hours | | G1-LB-12H | Percent total reads for GASright constructs grown in LB media, replicate 1 at 12 hours | | … | 
 
 | | G1-M9-30H | Percent total reads for GASright constructs grown in
 Maltose media, replicate 1 at 30 hours | | … | 
 
 | | L1-LB-0H | Percent total reads for Left constructs grown in LB media,
 replicate 1 at 0 hours | | R1-LB-0H | Percent total reads for Right
 constructs grown in LB media, replicate 1 at 0 hours | | … | 
 
 | ###### *Naming scheme:* G# = GASright replicate number\ L# = Left
 replicate number\ R# = Right replicate number\ LB = Luria Broth Growth
 sample\ M9 = Maltose Broth Growth sample\ \#H = # of hours of growth ##
 Contact Alessandro Senes, [senes@wisc.edu](mailto:senes@wisc.edu)