The results of CD measurements show that the studied N.HSV and C.HSV peptides tend to form a disordered structure in all tested conditions (water, DPC and POPC), while the transmembrane-cytoplasmic fragment of UL49.5 (TMC.HSV) forms an α-helical structure in DPC or POPC solutions. Comparing the CD and NMR spectra of the N-terminal peptide fragments of UL49.5 proteins from BoHV-1 and HSV-1 (current studies), it can be seen that the peptide forms a partially helical structure in BoHV-1 while unstructured in HSV-1. In addition, CD spectra shows of the BoHV-1-encoded N-terminal peptide becomes structured when binding to the cell membrane mimetic, while it is unstructured in the case of the HSV-1. To our knowledge, this is the first very clear difference in the spatial structure of the two proteins. CD studies of the transmembrane fragment of both proteins show that in the case of BoHV-1 the α-helix structure is formed at very low concentrations of POPC, while for the protein from HSV-1 the protein becomes structured during increasing concentrations of POPC. This means that it is likely that the transmembrane fragment of the BoHV-1-encoded UL49.5 protein has a stronger affinity for the membrane than that from HSV-1. This is also confirmed by our MD calculations, which show that the BoHV-1-encoded UL49.5 protein forms more contacts with lipid bilayer in total, tends to stay closer to lipid bilayer, and interacts with it than HSV-1-encoded UL49.5. This is an important difference in the behavior of the two proteins. The higher affinity of the BoHV-1-encoded UL49.5 protein to the ER membrane may, under natural conditions, result in improved membrane-incorporation, lower tendency to form aggregates or increased mobility in the ER membrane, facilitating the recognition of the TAP transporter. and in the supplementary materials. The sequential assignments were achieved by employing 2D homonuclear NOESY spectra. Additionally, we analyzed the chemical shifts of 15 N in the 1 H-15 N HSQC spectra under different concentrations of 5-DSA.