biostudies-literatureWebb KRNational Science Foundation Division of Graduate EducationNational Institutes of HealthVanderbilt UniversityNational Institute of General Medical SciencesNIGMS NIH HHSNational Science Foundation1491-1502https://www.ebi.ac.uk/biostudies/studies/S-EPMC10147839biostudies-literatureUnknown122(8)α-Helical secondary structures impart specific mechanical and physiochemical properties to peptides and proteins, enabling them to perform a vast array of molecular tasks ranging from membrane insertion to molecular allostery. Loss of α-helical content in specific regions can inhibit native protein function or induce new, potentially toxic, biological activities. Thus, identifying specific residues that exhibit loss or gain of helicity is critical for understanding the molecular basis of function. Two-dimensional infrared (2D IR) spectroscopy coupled with isotope labeling is capable of capturing detailed structural changes in polypeptides. Yet, questions remain regarding the inherent sensitivity of isotope-labeled modes to local changes in α-helicity, such as terminal fraying; the origin of spectral shifts (hydrogen-bonding versus vibrational coupling); and the ability to definitively detect coupled isotopic signals in the presence of overlapping side chains. Here, we address each of these points individually by characterizing a short, model α-helix (DPAEAAKAAAGR-NH₂) with 2D IR and isotope labeling. These results demonstrate that pairs of ¹³C¹⁸O probes placed three residues apart can detect subtle structural changes and variations along the length of the model peptide as the α-helicity is systematically tuned. Comparison of singly and doubly labeled peptides affirm that frequency shifts arise primarily from hydrogen-bonding, while vibrational coupling between paired isotopes leads to increased peak areas that can be clearly differentiated from underlying side-chain modes or uncoupled isotope labels not participating in helical structures. These results demonstrate that 2D IR in tandem with i,i+3 isotope-labeling schemes can capture residue-specific molecular interactions within a single turn of an α-helix.Biophysical journalProbing local changes to α-helical structures with 2D IR spectroscopy and isotope labeling.PMC101478395T32GM008320-321937963T32 GM0083201445197Webb KRShmidt ASegner KDBuchanan LEHess KAfalseProbing local changes to α-helical structures with 2D IR spectroscopy and isotope labeling.α-Helical secondary structures impart specific mechanical and physiochemical properties to peptides and proteins, enabling them to perform a vast array of molecular tasks ranging from membrane insertion to molecular allostery. Loss of α-helical content in specific regions can inhibit native protein function or induce new, potentially toxic, biological activities. Thus, identifying specific residues that exhibit loss or gain of helicity is critical for understanding the molecular basis of function. Two-dimensional infrared (2D IR) spectroscopy coupled with isotope labeling is capable of capturing detailed structural changes in polypeptides. Yet, questions remain regarding the inherent sensitivity of isotope-labeled modes to local changes in α-helicity, such as terminal fraying; the origin of spectral shifts (hydrogen-bonding versus vibrational coupling); and the ability to definitively detect coupled isotopic signals in the presence of overlapping side chains. Here, we address each of these points individually by characterizing a short, model α-helix (DPAEAAKAAAGR-NH₂) with 2D IR and isotope labeling. These results demonstrate that pairs of ¹³C¹⁸O probes placed three residues apart can detect subtle structural changes and variations along the length of the model peptide as the α-helicity is systematically tuned. Comparison of singly and doubly labeled peptides affirm that frequency shifts arise primarily from hydrogen-bonding, while vibrational coupling between paired isotopes leads to increased peak areas that can be clearly differentiated from underlying side-chain modes or uncoupled isotope labels not participating in helical structures. These results demonstrate that 2D IR in tandem with i,i+3 isotope-labeling schemes can capture residue-specific molecular interactions within a single turn of an α-helix.2023-01-01T00:00:00Z2023 Apr2024-11-13T12:29:08.363Z2024-11-13T12:29:08.363ZS-EPMC101478393690680010.1016/j.bpj.2023.03.014