Engineered Chimeras Unveil Swappable Modular Features of Fatty Acid and Polyketide Synthase Acyl Carrier Proteins.

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein, we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the Escherichia coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based cross-linking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step toward the strategic engineering of "un-natural" natural products.

Vibrational Spectroscopic Map, Vibrational Spectroscopy, and Intermolecular Interaction.

Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and interprotein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an all-encompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semiempirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository Web site (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-the-art multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.

Deuterium-Enhanced Raman Spectroscopy for Histidine pKa Determination in a pH-Responsive Hydrogel.

We report here a method for the determination of the pKa of histidine in complex or heterogeneous systems amenable to neither solid-state nor solution NMR spectroscopy. Careful synthesis of a fluorenylmethyloxycarbonyl- and trityl-protected, C2-deuterated histidine produces a vibrational-probe-equipped amino acid that can readily be incorporated into any peptide accessible by standard solid-phase methods. The frequency of the unique, Raman-active stretching vibration of this C2-D probe is a clear reporter of the protonation state of histidine. We investigate here a pH-sensitive peptide that self-assembles to form a hydrogel at neutral pH. The pKa of the lone histidine residue in the peptide, which is likely responsible for this pH-dependent behavior, cannot be investigated by NMR spectroscopy because of the supramolecular, soft nature of the gel. However, after synthesizing a C2-deuterated-histidine-containing peptide, we were able to follow the protonation state of histidine throughout a pH titration using Raman difference spectroscopy, thereby precisely determining the pKa of interest.

pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy.

Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, ¹H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine's imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via ¹H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa's of histidine-containing peptide and protein systems.

Cyanylated Cysteine Reports Site-Specific Changes at Protein-Protein-Binding Interfaces Without Perturbation.

To investigate the cyanylated cysteine vibrational probe group's ability to report on binding-induced changes along a protein-protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques.

Vibrational Probes: From Small Molecule Solvatochromism Theory and Experiments to Applications in Complex Systems.

The vibrational frequency of a chosen normal mode is one of the most accurately measurable spectroscopic properties of molecules in condensed phases. Accordingly, infrared absorption and Raman scattering spectroscopy have provided valuable information on both distributions and ensemble-average values of molecular vibrational frequencies, and these frequencies are now routinely used to investigate structure, conformation, and even absolute configuration of chemical and biological molecules of interest. Recent advancements in coherent time-domain nonlinear vibrational spectroscopy have allowed the study of heterogeneous distributions of local structures and thermally driven ultrafast fluctuations of vibrational frequencies. To fully utilize IR probe functional groups for quantitative bioassays, a variety of biological and chemical techniques have been developed to site-specifically introduce vibrational probe groups into proteins and nucleic acids. These IR-probe-labeled biomolecules and chemically reactive systems are subject to linear and nonlinear vibrational spectroscopic investigations and provide information on the local electric field, conformational changes, site-site protein contacts, and/or function-defining features of biomolecules. A rapidly expanding library of data from such experiments requires an interpretive method with atom-level chemical accuracy. However, despite prolonged efforts to develop an all-encompassing theory for describing vibrational solvatochromism and electrochromism as well as dynamic fluctuations of instantaneous vibrational frequencies, purely empirical and highly approximate theoretical models have often been used to interpret experimental results. They are, in many cases, based on the simple assumption that the vibrational frequency of an IR reporter is solely dictated by electric potential or field distribution around the vibrational chromophore. Such simplified description of vibrational solvatochromism generally referred to as vibrational Stark effect theory has been considered to be quite appealing and, even in some cases, e.g., carbonyl stretch modes in amide, ester, ketone, and carbonate compounds or proteins, it works quantitatively well, which makes it highly useful in determining the strength of local electric field around the IR chromophore. However, noting that the vibrational frequency shift results from changes of solute-solvent intermolecular interaction potential along its normal coordinate, Pauli exclusion repulsion, polarization, charge transfer, and dispersion interactions, in addition to the electrostatic interaction between distributed charges of both vibrational chromophore and solvent molecules, are to be properly included in the theoretical description of vibrational solvatochromism. Since the electrostatic and nonelectrostatic intermolecular interaction components have distinctively different distance and orientation dependences, they affect the solvatochromic vibrational properties in a completely different manner. Over the past few years, we have developed a systematic approach to simulating vibrational solvatochromic data based on the effective fragment potential approach, one of the most accurate and rigorous theories on intermolecular interactions. We have further elucidated the interplay of local electric field with the general vibrational solvatochromism of small IR probes in either solvents or complicated biological systems, with emphasis on contributions from non-Coulombic intermolecular interactions to vibrational frequency shifts and fluctuations. With its rigorous foundation and close relation to quantitative interpretation of experimental data, this and related theoretical approaches and experiments will be of use in studying and quantifying the structure and dynamics of biomolecules with unprecedented time and spatial resolution when combined with time-resolved vibrational spectroscopy and chemically sensitive vibrational imaging techniques.

Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups.

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein-protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes' local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin's variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

Acyl Carrier Protein Cyanylation Delivers a Ketoacyl Synthase-Carrier Protein Cross-Link.

Acyl carrier proteins (ACPs) are central hubs in polyketide and fatty acid biosynthetic pathways, but the fast motions of the ACP's phosphopantetheine (Ppant) arm make its conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we report that converting the terminal thiol of Escherichia coli ACP's Ppant arm into a thiocyanate activates this site to form a selective cross-link with the active site cysteine of its partner ketoacyl synthase (FabF). The reaction releases a cyanide anion, which can be detected by infrared spectroscopy. This represents a practical and generalizable method for obtaining and visualizing ACP-protein complexes relevant to biocatalysis and will be valuable in future structural and engineering studies.

Dynamic asymmetry and the role of the conserved active-site thiol in rabbit muscle creatine kinase.

Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multipopulation CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared line shape. Together, these spectral observations localized at the active-site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.

Probing the phosphopantetheine arm conformations of acyl carrier proteins using vibrational spectroscopy.

Acyl carrier proteins (ACPs) are universal and highly conserved domains central to both fatty acid and polyketide biosynthesis. These proteins tether reactive acyl intermediates with a swinging 4'-phosphopantetheine (Ppant) arm and interact with a suite of catalytic partners during chain transport and elongation while stabilizing the growing chain throughout the biosynthetic pathway. The flexible nature of the Ppant arm and the transient nature of ACP-enzyme interactions impose a major obstacle to obtaining structural information relevant to understanding polyketide and fatty acid biosynthesis. To overcome this challenge, we installed a thiocyanate vibrational spectroscopic probe on the terminal thiol of the ACP Ppant arm. This site-specific probe successfully reported on the local environment of the Ppant arm of two ACPs previously characterized by solution NMR, and was used to determine the solution exposure of the Ppant arm of an ACP from 6-deoxyerythronolide B synthase (DEBS). Given the sensitivity of the probe's CN stretching band to conformational distributions resolved on the picosecond time scale, this work lays a foundation for observing the dynamic action-related structural changes of ACPs using vibrational spectroscopy.

A new Raman spectroscopic probe of both the protonation state and noncovalent interactions of histidine residues.

The amino acid histidine (His) has a number of unique roles that can dictate function in proteins, and these roles are typically conferred through noncovalent interactions that depend on the protonation state of His's 4-substituted imidazole ring. His's protonation state can vary near physiological pH, and a probe of His's variable protonation state and its resulting noncovalent interactions that has both high time resolution and no sample limitations could find wide use in determining the role of particular His residues in proteins. Here we use a classic deuterium exchange reaction to replace the C2-H hydrogen atom of the His imidazole ring with deuterium, leading to a unique aromatic C2-D stretching vibration whose frequency is sensitive to environmental changes across the entire imidazole ring. Using nonresonant Raman spectroscopy, we demonstrate using model compounds that the frequency of this C2-D vibration shifts by 35 cm(-1) upon changes in the His protonation state. The C2-D band is a very weak infrared absorber, so this vibration is not expected to be useful in infrared transmission experiments for proteins. Solvent-dependent Raman experiments indicate that the C2-D band of the neutral imidazole ring is sensitive to H-bonding interaction with donors and acceptors of varying strengths, suggesting that the C2-D frequency can be used to identify H-bonding partners of specific His residues. Raman spectra at varying concentrations of Cu(2+) also show the C2-D band's sensitivity to metal coordination, with differences due to changes in the coordination environment. The strong Raman signal of this band and the sampling flexibility of Raman spectroscopy suggest that this vibration could be very useful in documenting the local role of His residues in many His-containing proteins and protein assemblies.

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.

Heterologous Expression, Purification, and Characterization of Type II Polyketide Synthase Acyl Carrier Proteins.

The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs.

Using infrared spectroscopy of cyanylated cysteine to map the membrane binding structure and orientation of the hybrid antimicrobial peptide CM15.

The synthetic antimicrobial peptide CM15, a hybrid of N-terminal sequences from cecropin and melittin peptides, has been shown to be extremely potent. Its mechanism of action has been thought to involve pore formation based on prior site-directed spin labeling studies. This study examines four single-site ß-thiocyanatoalanine variants of CM15 in which the artificial amino acid side chain acts as a vibrational reporter of its local environment through the frequency and line shape of the unique CN stretching band in the infrared spectrum. Circular dichroism experiments indicate that the placements of the artificial side chain have only small perturbative effects on the membrane-bound secondary structure of the CM15 peptide. All variant peptides were placed in buffer solution, in contact with dodecylphosphatidylcholine micelles, and in contact with vesicles formed from Escherichia coli polar lipid extract. At each site, the CN stretching band reports a different behavior. Time-dependent attenuated total reflectance infrared spectra were also collected for each variant as it was allowed to remodel the E. coli lipid vesicles. The results of these experiments agree with the previously proposed formation of toroidal pores, in which each peptide finds itself in an increasingly homogeneous and curved local environment without apparent peptide-peptide interactions. This work also demonstrates the excellent sensitivity of the SCN stretching vibration to small changes in the peptide-lipid interfacial structure.

Probing structural transitions in the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by vibrational spectroscopy of cyanylated cysteines.

Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) were cyanylated at cysteine and their infrared spectra in the C triple bond N stretching region were recorded both in the absence and in the presence of one of the physiological partners of N(TAIL), namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within N(TAIL), the C triple bond N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level.

Tracking carrier protein motions with Raman spectroscopy.

Engineering microbial biosynthetic pathways represents a compelling route to gain access to expanded chemical diversity. Carrier proteins (CPs) play a central role in biosynthesis, but the fast motions of CPs make their conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we present a low-resource method to directly reveal carrier protein-substrate interactions. Chemoenzymatic loading of commercially available, alkyne-containing substrates onto CPs enables rapid visualization of the molecular cargo's local environment using Raman spectroscopy. This method could clarify the foundations of the chain sequestration mechanism, facilitate the rapid characterization of CPs, and enable visualization of the vectoral processing of natural products both in vitro and in vivo.

A Direct, Quantitative Connection between Molecular Dynamics Simulations and Vibrational Probe Line Shapes.

A quantitative connection between molecular dynamics simulations and vibrational spectroscopy of probe-labeled systems would enable direct translation of experimental data into structural and dynamical information. To constitute this connection, all-atom molecular dynamics (MD) simulations were performed for two SCN probe sites (solvent-exposed and buried) in a calmodulin-target peptide complex. Two frequency calculation approaches with substantial nonelectrostatic components, a quantum mechanics/molecular mechanics (QM/MM)-based technique and a solvatochromic fragment potential (SolEFP) approach, were used to simulate the infrared probe line shapes. While QM/MM results disagreed with experiment, SolEFP results matched experimental frequencies and line shapes and revealed the physical and dynamic bases for the observed spectroscopic behavior. The main determinant of the CN probe frequency is the exchange repulsion between the probe and its local structural neighbors, and there is a clear dynamic explanation for the relatively broad probe line shape observed at the "buried" probe site. This methodology should be widely applicable to vibrational probes in many environments.

The Bend+Libration Combination Band Is an Intrinsic, Collective, and Strongly Solute-Dependent Reporter on the Hydrogen Bonding Network of Liquid Water.

Water is an extensively self-associated liquid due to its extensive hydrogen bond (H-bond) forming ability. The resulting H-bonded network fluid exhibits nearly continuous absorption of light from the terahertz to the near-IR region. The relatively weak bend+libration water combination band (centered at 2130 cm-1) has been largely overlooked as a reporter of liquid water's structure and dynamics despite its location in a convenient region of the IR for spectroscopic study. The intermolecular nature of the combination band leads to a unique absorption signal that reports collectively on the rigidity of the H-bonding network in the presence of many different solutes. This study reports comprehensively how the combination band acts as an intrinsic and collective probe in various chemically and biologically relevant solutions, including salts of varying character, denaturants, osmolytes, crowders, and surfactants that form reverse micelles and micelles. While we remark on changes in the line width and intensity of this combination band, we mainly focus on the frequency and how the frequency reports on the collective H-bonding network of liquid water. We also comment on the "association band" moniker often applied to this band and how to evaluate discrete features in this spectral region that sometimes appear in the IR spectra of specific kinds of aqueous samples of organic solutes, especially those with very high solute concentrations, with the conclusion that most of these discrete spectral features come exclusively from the solutes and do not report on the water. Contrasts are drawn throughout this work between the collective and delocalized reporting ability of the combination band and the response of more site-specific vibrations like the much-investigated OD stretch of HDO in H2O: the combination band is a unique reporter of H-bonding structure and dynamics and fundamentally different than any local mode probe. Since this band appears as the spectroscopic "background" for many local-mode reporter groups, we note the possibility of observing both local and collective solvent dynamics at the same time in this spectral region.

Covalently bound azido groups are very specific water sensors, even in hydrogen-bonding environments.

Covalently bound azido groups are found in many commercially available biomolecular precursors and substrates, and the NNN asymmetric stretching band of these groups is a strong infrared absorber that appears in a spectral region clear of other signals. In order to evaluate comprehensively the solvatochromism of the asymmetric azido NNN stretching band for site-specific use in biomolecular contexts, infrared spectra of the model compounds 5-azido,1-pentanoic acid and 3-(p-azidophenyl),1-propanoic acid were acquired in a large variety of nonpolar, polar, and hydrogen-bond-donating solvents, as well as mixed aqueous-organic solvents. Spectra in pure solvents indicated that the aliphatic NNN stretching frequency maximum does not depend on solvent polarity, while the aromatic NNN frequency displays a weak but nonzero sensitivity to polarity. In both cases, the NNN frequency exhibits a blue-shift in H-bond-donating solvents, but the frequency in water is higher than in any other H-bond-donating solvent including solvents that are stronger H-bond donors. In nonfluorinated H-bond donor solvents, the frequency blue shift scales with the density of H-bond donors. This sensitivity to the presence of water was further explored in several mixed solvent environments, with the conclusion that this vibrational mode is a highly specific sensor of hydration, even in environments containing other H-bond donors like amides and alcohols, due to the very high local density of H-bond donors in water. The relatively uncomplicated (compared to nitriles, for example), water-specific response of this vibrational mode should lead to its adoption as a site-specific probe of hydration in many different possible systems in which the presence and role of molecular water is of primary interest.