Frequency Changes in Terminal Alkynes Provide Strong, Sensitive, and Solvatochromic Raman Probes of Biochemical Environments.

The C≡C stretching frequencies of terminal alkynes appear in the "clear" window of vibrational spectra, so they are attractive and increasingly popular as site-specific probes in complicated biological systems like proteins, cells, and tissues. In this work, we collected infrared (IR) absorption and Raman scattering spectra of model compounds, artificial amino acids, and model proteins that contain terminal alkyne groups, and we used our results to draw conclusions about the signal strength and sensitivity to the local environment of both aliphatic and aromatic terminal alkyne C≡C stretching bands. While the IR bands of alkynyl model compounds displayed surprisingly broad solvatochromism, their absorptions were weak enough that alkynes can be ruled out as effective IR probes. The same solvatochromism was observed in model compounds' Raman spectra, and comparisons to published empirical solvent scales (including a linear regression against four meta-aggregated solvent parameters) suggested that the alkyne C≡C stretching frequency mainly reports on local electronic interactions (i.e., short-range electron donor-acceptor interactions) with solvent molecules and neighboring functional groups. The strong solvatochromism observed here for alkyne stretching bands introduces an important consideration for Raman imaging studies based on these signals. Raman signals for alkynes (especially those that are π-conjugated) can be exceptionally strong and should permit alkynyl Raman signals to function as probes at very low concentrations, as compared to other widely used vibrational probe groups like azides and nitriles. We incorporated homopropargyl glycine into a transmembrane helical peptide via peptide synthesis, and we installed p-ethynylphenylalanine into the interior of the Escherichia coli fatty acid acyl carrier protein using a genetic code expansion technique. The Raman spectra from each of these test systems indicate that alkynyl C≡C bands can act as effective and unique probes of their local biomolecular environments. We provide guidance for the best possible future uses of alkynes as solvatochromic Raman probes, and while empirical explanations of the alkyne solvatochromism are offered, open questions about its physical basis are enunciated.

The Effect of Divalent Cations on the Thermostability of Type II Polyketide Synthase Acyl Carrier Proteins.

The successful engineering of biosynthetic pathways hinges on understanding the factors that influence acyl carrier protein (ACP) stability and function. The stability and structure of ACPs can be influenced by the presence of divalent cations, but how this relates to primary sequence remains poorly understood. As part of a course-based undergraduate research experience, we investigated the thermostability of type II polyketide synthase (PKS) ACPs. We observed an approximate 40 °C range in the thermostability amongst the 14 ACPs studied, as well as an increase in stability (5 - 26 °C) of the ACPs in the presence of divalent cations. Distribution of charges in the helix II-loop-helix III region was found to impact the enthalpy of denaturation. Taken together, our results reveal clues as to how the sequence of type II PKS ACPs relates to their structural stability, information that can be used to study how ACP sequence relates to function.