Chapter 18 Beyond Supersecondary Structure: Physics-Based Sequence Alignment S. Rackovsky Abstract Traditional approaches to sequence alignment are based on evolutionary ideas. As a result, they are prebiased toward results which are in accord with initial expectations. We present here a method of sequence alignment which is based entirely on the physical properties of the amino acids. This approach has no inherent bias, eliminates much of the computational complexity associated with methods currently in use, and has been shown to give good results for structures which were poorly predicted by traditional methods in recent CASP competitions and to identify sequence differences which correlate with structural and dynamic differences not detectable by traditional methods. Key words Sequence alignment, Amino acid physical properties, Physics-based alignment, Evolution- free alignment, Homology modeling, Protein structure prediction 1 Introduction Protein sequence alignment is one of the most commonly applied techniques in modern molecular biology. There are two circum- stances in which sequence alignment is useful: (a) The establishment of an evolutionary relationship between sequences of interest. (b) The establishment of a structural relationship between sequences of interest. It has been recognized since the earliest days of protein science [1] that, in organisms which are evolutionarily related, corresponding proteins will exhibit similar sequences and evolu- tionary clocks have been constructed based on the analysis of that sequence similarity. It is also a fundamental tenet of protein structure studies, as currently practiced, that similarity between the structures of two proteins arises from similarity between their sequences. This is the basis for homology modeling [2–4], which remains an important Alexander E. Kister (ed.), Protein Supersecondary Structures: Methods and Protocols, Methods in Molecular Biology, vol. 1958, https://doi.org/10.1007/978-1-4939-9161-7_18, © Springer Science+Business Media, LLC, part of Springer Nature 2019 341