The prediction accuracy for true positives and negatives is reported to be close to 100%, and the remaining main cause of false positive prediction is hydrophobic α-helices completely buried in the hydrophobic core of proteins. These biological dispositions are exploitable in terms of transmembrane region prediction in query protein sequences, and tools such as the quite reliable TMHMM (software for predicting TMHs based on a hidden Markov model), Phobius or the dense alignment surface-transmembrane filter (DAS-TMfilter) represent today’s prediction limit of TMHs’ hydrophobic cores within the protein sequence. These properties vary somewhat in length and intensity between various biological organelle membranes, between prokaryotes and eukaryotes and even amongst eukaryotic species studied due to slightly different membrane constraints. Besides the span of generally hydrophobic residues in the TMH, there are other trends in the sequence such as a saddle-like distribution of polar residues (depressed incidence of charged residues in the TMH itself), an enriched occurrence of positively charged residues in the cytosolic flanking regions as well as an increased likelihood of tryptophan and tyrosine at either flank edge. The relationship between sequence patterns in and in the vicinity of TMHs and their structural and functional properties, as well as their interaction with the lipid bilayer membrane, has been a field of intensive research in the last three decades. As these additional biological functions are mirrored in the TMHs’ sequence patterns, TMHs can be classified as simple (just hydrophobic anchors) and complex sequence segments. From the biological function point of view, many TMHs have multiple roles besides being just hydrophobic anchors for example, certain TMHs have been identified as regulators of protein quality control and trafficking mechanisms. The insertion and formation of the transmembrane helices (TMHs) follow a complex thermodynamic equilibrium. They are capable of just partially spanning the membrane, spanning using oblique angles, and even lying flat on the membrane surface. Although this is broadly true, hundreds of high-quality membrane structures have elucidated that membrane-embedded helices can adopt a plethora of lengths and orientations within the membrane. Two decades ago, the classic concept of a transmembrane helical region was a rather simple story: Typical transmembrane proteins were thought to be anchored in the membrane by membrane-spanning bundles of non-polar α-helices of roughly 20 residues in length, with a consistent orientation of being perpendicular to the membrane surface. These observations will stimulate new prediction methods for TMHs and protein topology from a sequence as well as new engineering designs for artificial membrane proteins.

The charged-residue flank bias is only one of the TMH sequence features with a role in the anchorage mechanisms, others apparently being the leucine intra-helix propensity skew towards the cytoplasmic side, tryptophan flanking as well as the cysteine and tyrosine inside preference. The effect is most pronounced for TMHs from single-pass transmembrane (bitopic) proteins compared to those with multiple TMHs (polytopic proteins) and especially for the class of simple TMHs that evolved for the sole role as membrane anchors. We observe variations amongst taxa as well as for organelles along the secretory pathway. As negative residues are generally rare within or near TMHs, the statistical significance is sensitive with regard to details of TMH alignment and residue frequency normalisation and also to dataset size therefore, this trend was obscured in previous work. With statistics derived from a large body of protein sequences, we demonstrate that, in addition to the positive charge preference at the cytoplasmic inside (positive-inside rule), negatively charged residues preferentially occur or are even enriched at the non-cytoplasmic flank or, at least, they are suppressed at the cytoplasmic flank (negative-not-inside/negative-outside (NNI/NO) rule). Physical constraints such as the membrane’s hydrophobicity and electrostatic potential apply uniform requirements to TMHs and their flanking regions consequently, they are mirrored in their sequence patterns (in addition to TMHs being a span of generally hydrophobic residues) on top of variations enforced by the specific protein’s biological functions. Transmembrane helices (TMHs) frequently occur amongst protein architectures as means for proteins to attach to or embed into biological membranes.

Multipass transmembrane protein