Temperature-dependent FTIR Spectroscopy Research Articles

Dynamic Multicomponent Reactions-Directed Self-Assembled G-quadruplex Inherent Antibacterial Hydrogel.

Nowadays, inherent antibacterial hydrogels have gained significant attention due to their utilization against infectious bacteria. Herein, we focus on the development of an injectable, self-healable, dynamic, and G-quadruplex hydrogel with inherent antibacterial activity. The dynamic self-assembled hydrogel is constructed upon multicomponent reactions (MCR) among guanosine, 2-formylphenylboronic acid, and amino acid/peptides in the presence of potassium ions. The role of amino acid/peptides in the formation of the G-quadruplex hydrogel is studied in detail. The G-quadruplex structure is formed via the π-π stacking of G-quartets. The formation of G-quadruplex is investigated by thioflavin T binding assay, CD spectroscopy, and PXRD. The formation of the dynamic imino-boronate bond in the hydrogels is well characterized by temperature-dependent 11B NMR (VT-NMR) and FT-IR spectroscopy. Furthermore, HR-TEM images and rheological experiments reveal the fibrillar networks and viscoelastic property of the hydrogels. The presence of the dynamic imino-boronate ester bonds makes the hydrogel injectable and self-healable in nature. These dynamic G-quadruplex hydrogels show potential antibacterial activity against a series of Gram-positive and Gram-negative bacteria. The hydrogels have been used for the entrapment and sustained release of an anticancer drug doxorubicin over 48 h at different pHs (4.8, 7.4, and 8.5) and temperature without the influence of any external stimuli. Such injectable and self-healable hydrogels could be used in various applications in the field of biomedical science.

Water does not dance as ions sing: A new approach in elucidation of ion-invariant water fluctuations

Aqueous solutions of salts composed from monovalent ions are explored using temperature-dependent FT-IR spectroscopy in transmission. Water combination band, being extremely sensitive to the network of hydrogen bonds due to the contribution of water librations (ρLH2O), is analyzed in uni- and multivariate fashion. Univariate analysis of the combination band maximum (νmax) reveals that perturbation of water hydrogen bond network by ions is primary driven by electrostatic interactions between water and ions. Using multivariate curve resolution with alternating least squares and evolving factor analysis this band is separated into two components that represent low- and high-density water. The observed asymmetry in their behavior is interpreted in terms of fluctuations of a hydrogen bond network of two water components. The significance of the found phenomenon is unambiguously confirmed by performing analogous analysis in the spectral range that contains partial signature of water linear bending (δHOH) and is free from ρLH2O, in which the asymmetry is absent. Additionally, we show that this phenomenon, namely ion-invariant behavior of water fluctuations, persists even in the regime of high ionic strengths. Although ions indeed participate in shaping of water hydrogen bond network, this straightforward approach shows that its temperature-dependent fluctuations are ion-independent.

Application of MCR-ALS with EFA on FT-IR spectra of lipid bilayers in the assessment of phase transition temperatures: Potential for discernment of coupled events

Temperature-dependent transmission FT-IR spectroscopy and DSC measurements were conducted on lipid multibilayers constituted from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Lipid multibilayers made from 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, which do not form a ripple phase, were examined as a reference. Spectra were analyzed using multivariate curve resolution technique with alternating least squares and evolving factor analysis (MCR-ALS with EFA) and lipid phase transition temperatures were determined. Polar parts of lipid molecules exert greater response on a ripple phase formation than non-polar ones. However, vibrational signatures of hydrocarbon chains with intramolecular origins display certain qualitative differences that pave the way for future work oriented on uncoupling the events that drive ripple phase formation.

Tailored Crosslinking Process and Protective Efficiency of Epoxy Coatings Containing Glycidyl-POSS.

Versatile product protective coatings that deliver faster drying times and shorter minimum overcoat intervals that enable curing at faster line speeds and though lower energy consumption are often desired by coating manufacturers. Product protective coatings, based on silsesquioxane-modified diglycidyl ether of bisphenol-A (DGEBA) epoxy resin, are prepared through a glycidyl ring-opening polymerization using dicyandiamide (DICY) as a curing agent. As silsesquioxane modifier serves the octaglycidyl-polyhedral oligomeric silsesquioxane (GlyPOSS). To decrease the operational temperature of the curing processes, three different accelerators for crosslinking are tested, i.e., N,N-benzyl dimethylamine, 2-methylimidazole, and commercial Curezol 2MZ-A. Differential scanning calorimetry, temperature-dependent FT-IR spectroscopy, and rheology allow differentiation among accelerators’ effectiveness according to their structure. The former only contributed to epoxy ring-opening, while the latter two, besides participate in crosslinking. The surface roughness of the protective coatings on aluminum alloy substrate decreases when the accelerators are applied. The scanning electron microscopy (SEM) confirms that coatings with accelerators are more homogeneous. The protective efficiency is tested with a potentiodynamic polarization technique in 0.5 M NaCl electrolyte. All coatings containing GlyPOSS, either without or with accelerators, reveal superior protective efficiency compared to neat DGEBA/DICY coating.

Swelling behaviour of core-shell microgels in H2O, analysed by temperature-dependent FTIR spectroscopy.

Stimuli-responsive microgels are colloidal particles and promising candidates for applications such as targeted drug delivery, matrices for catalysts, nanoactuators and smart surface coatings. To tailor the response, the architecture of microgels is of paramount importance with respect to these applications. Statistical copolymer microgels based on N-isopropylmethacrylamide (NiPMAM) and N-n-propylacrylamide (NnPAM) show a cooperative phase transition leading to a collapse at a specific temperature. Interestingly, some core-shell microgel particles reveal a linear response of the hydrodynamic radius with temperature. Such observations were made by photon correlation spectroscopy (PCS), which is limited to the diffusion properties dominated by the particle shell. In this work we investigate the molecular hydration within the network of microgels in H2O by temperature-dependent FTIR spectroscopy. The phase transition temperature was determined by the shift in frequency of the NH bending vibration in homopolymer and statistical copolymer microgels and the results are in accordance with those from PCS. In contrast, experiments on core-shell particles show a broadening and shift of the respective phase transition temperatures of the core and shell indicating an interaction of the core and shell polymers on a molecular level that extends far into the core. In conclusion, temperature-dependent FTIR spectroscopy is a convenient approach to elucidate the internal architecture of complex microgel particles in H2O.

Supramolecular Networks from Block Copolymers Based on Styrene and Isoprene Using Hydrogen Bonding Motifs-Part 1: Synthesis and Characterization.

The combination of controlled anionic polymerization and subsequent introduction of hydrogen bonding groups was established to form thermo-reversible, supramolecular networks. Several polyisoprene-block-polystyrene-block-polyisoprene (ISI) copolymers—with polystyrene (PS) as the main block, and consequently giving the decisive material characteristics—were synthesized. The novel modification approach to post-functionalize the polyisoprene (PI) end-blocks and to introduce different motifs, which are able to form self-complementary hydrogen bonds, was attained. In the first step, hydroxylation was accomplished using 9-borabicyclo[3.3.1]nonane. Starting from the hydroxylated polymer, esterification with succinic anhydride was implemented to form an ester group with carboxylic end-group (-O-CO-CH2-CH2-COOH). In a second approach, 1,1’-carbonyldiimidazole was used as coupling agent to introduce various types of diamines (diethylenetriamine, triethylentetramine, and 2,6-diaminopyridine) to prepare urethane groups with amine end-group (-O-CO-NH-R-NH2). 1H NMR spectroscopy was used to confirm the successful synthesis and to calculate the degree of functionalization Df. Differential scanning calorimetry (DSC) showed a difference of the glass transition temperature Tg between unfunctionalized and functionalized block copolymers, but no greater influence between the different types of modification, and thus, on the Tg of the PS block. In temperature dependent FTIR spectroscopy, reversible processes were observed.

Online tracking of the thermal reduction of graphene oxide by two-dimensional correlation infrared spectroscopy

Thermal reduction of graphene oxide (GO) via rapid heating is an environment-friendly and cost-effective method. However, the detailed reduction mechanism remains unclear because of lack of methods for online monitoring of GO thermal reduction. In this study, the thermal reduction of GO (from 20 °C up to 400 °C in argon atmosphere) was successfully monitored online and investigated through temperature-dependent FTIR spectroscopy combined with scaling-MW2D FTIR spectroscopy and generalized 2D correlation analyses. Raman spectroscopy, XPS, and XRD studies were also conducted to characterize the structural changes before and after reduction. SEM and AFM analyses were performed to directly observe the formation of defects in GO after thermal reduction. According to scaling-MW2D results, the thermal reduction of GO was divided into two processes, namely, 35 °C–182 °C (process I) and 182 °C–385 °C (process II). Process I rapidly eliminates oxygen functional groups, and process II gradual removes them. The 2D correlation analysis for each process indicated the sequential order of movements of the oxygen-containing functional groups during thermal reduction. Process I comprised six steps, and process II contained four sequential steps. This work elucidated the complex deoxygenation steps and the mechanism of GO thermal reduction.

Interpreting Quasi-Thermal Effects in Ultrafast Spectroscopy of Hydrogen-Bonded Systems.

Vibrational excitation of molecules in the condensed phase relaxes through vibrational modes of decreasing energy to ultimately generate an equilibrium state in which the energy is distributed among low-frequency modes. In ultrafast vibrational spectroscopy, changes in the vibrational features of hydrogen-bonded NH and OH stretch modes are typically observed to persist long after these high-frequency vibrations have relaxed. Due to the resemblance to the spectral changes caused by heating the sample, these features are typically described as arising from a hot ground state. However, these spectral features appear on ultrafast time scales that are much too fast to result from a true thermal state, and significant differences between the thermal difference spectrum and the induced quasi-thermal changes in ultrafast spectroscopy are often observed. Here, we examine and directly compare the thermal and quasi-thermal responses of the hydrogen-bonded homodimer of 7-azaindole with temperature-dependent FTIR spectroscopy and ultrafast mid-IR continuum spectroscopy. We find that the thermal difference spectra contain contributions from both dissociation of the hydrogen bonds and from frequency shifts due to changes in the thermal population of low-frequency modes. The transient spectra in ultrafast vibrational spectroscopy are also found to contain two contributions: initial frequency shifts over 2.3 ± 0.11 ps associated with equilibration of the initial excitation, and frequency shifts associated with the excitation of several fingerprint modes, which decay over 21.8 ± 0.11 ps, giving rise to a quasi-thermal response caused by a distribution of fingerprint modes being excited within the sample ensemble. This resembles the thermal frequency shifts due to population changes of low-frequency modes, but not the overall thermal spectrum, which is dominated by features caused by dimer dissociation. These findings provide insight into the changes in the vibrational spectrum from different origins and are important for assigning, analyzing, and comparing features in thermal and ultrafast vibrational spectroscopy of hydrogen-bonded complexes.

Composite anion exchange membranes with functionalized hydrophilic or hydrophobic titanium dioxide

Composite anion exchange membranes based on polysulfone with grafted 1,4-diazabicyclo[2.2.2]octane (DABCO) and surface functionalized TiO2 nanoparticles were prepared and characterized. Tri(hydroxymethyl)propane (TMP) was used for hydrophilic and polymethyl-hydrosiloxane (PMHS) for hydrophobic surface functionalization. Thermal analysis (TGA and DSC) showed water loss, the loss of quaternary ammonium groups around 300 °C and the backbone decomposition above 400 °C starting from the quaternary carbon. The decompositions were confirmed by temperature-dependent FTIR spectroscopy. The thermomechanical study (DMA) showed a secondary relaxation, probably due to DABCO side groups motions, around 130 °C followed by the glass transition around 250 °C and by a partial crystallization of the polymer, which is also observed by DSC. The increase of storage modulus observed for the composites is consistent with their reduced hydration, which is confirmed by water uptake measurements. Atomic Force Microscopy observations show a more homogeneous dispersion of hydrophobic TiO2 nanoparticles, which are also well embedded in the polymer, whereas agglomeration is evident for hydrophilic surface treatment. The contact angle with water decreases strongly in composites with TMP-TiO2. The ionic conductivity is higher for PMHS-TiO2, probably related to the more homogeneous filler distribution with lower agglomeration.

Micro-dynamics mechanism of the phase transition behavior of poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate) hydrogels revealed by two-dimensional correlation spectroscopy

The micro-dynamics mechanism of the volume phase transition of PNIPAM-co-HEMA hydrogels was established using temperature-dependent FTIR spectroscopy, PCMW2D, and 2DCOS analysis.

Dehydration of α-oxalic acid dihydrate: Structural, spectroscopic and thermal study with implications on the disruption of water molecular bridges in soil organic matter

Water molecule bridges (WaMB), representing linkages between functional groups in organic matter, are hypothesized to stabilize the supramolecular structure of the soil organic matter (SOM). As the underlying mechanisms are largely unknown, partly due to the high heterogeneity, complexity and amorphicity in SOM, a more general understanding on the processes potentially underlying WaMB formation and disruption can be accomplished by exploring a simpler model system. We examined in a first step and for the first time the mechanism of water liberation in a well-defined model system, α-oxalic acid dihydrate, using XRPD, FT-IR, DSC and solid state 1H NMR. In a second step, we evaluated the potential of the transfer of this knowledge to the processes occurring in SOM. According to XRPD, dehydration occurs at about 70°C and it is accompanied by a change in the crystal system from monoclinic to orthorhombic. Temperature-dependent FT-IR spectra, apart from confirming the findings of XRPD, reveal the changes in hydrogen bonding pattern upon transformation of the dihydrate to the anhydrous compound. DSC thermograms obtained in two subsequent heating and cooling runs suggest a reversibility and heating-rate independence in breaking and re-forming of hydrogen bonds between oxalic acid moieties and water molecules in temperature range t*∼60–76°C. The latter is also confirmed by solid state 1H NMR spectra. Similarly to α-oxalic acid dihydrate, disruption of WaMB in SOM occurs in that temperature range, but does not reappear immediately in the second heating run and it is heating-rate dependent, which points to a relevance of structural relaxation processes in SOM. Simultaneous monitoring of spectral signatures changes and detection of the water release temperature suggests that temperature-dependent FT-IR spectroscopy might be a helpful method to monitor WaMB disruption and formation in SOM.

The role of hydrogen bonding in the phase behaviour of supramolecular liquid crystal dimers

The phase behaviour of equimolar mixtures of 4-octyloxybenzoic acid, OOBA, and either 1-(4-butylazobenzene-4′-oxy)-6-(4-oxypyridine)pentane, BuABO5OPyr, or 1-(4-butylazobenzene-4′-oxy)-6-(4-oxypyridine)hexane, BuABO6OPyr, is reported. OOBA exhibits enantiotropic smectic C and nematic phases, whereas BuABO5OPyr and BuABO6OPyr are not liquid crystalline. Both the OOBA/BuABO5OPyr and OOBA/BuABO6OPyr equimolar mixtures show an enantiotropic smectic A phase identified using X-ray diffraction and polarised light microscopy. The liquid crystallinty of the mixtures is attributed, in part, to the formation of a 1:1 hydrogen-bonded complex between OOBA and either BuABO5OPyr or BuABO6OPyr. Differences in the transitional properties of the mixtures are accounted for in terms of the average shapes of these 1:1 complexes. Temperature dependent FTIR spectroscopy shows, however, that the formation of the complex is not quantitative in the mixtures but instead a complex mixture exists over the complete temperature range studied, involving the 1:1 complex, both cyclic and open acid dimers, free acid and hence, free BuABOnOPyr molecules.

New insights into the liquid crystal behaviour of hydrogen-bonded mixtures provided by temperature-dependent FTIR spectroscopy

The phase behaviour of equimolar mixtures of 6-(4ʹ-cyanobiphenyl-4-yl)hexyloxybenzoic acid (CB6OBA) with either 1-(4-butylazobenzene-4ʹ-oxy)-5-(4-oxypyridine)pentane (BuABO5OPyr) or 1-(4-butylazobenzene-4ʹ-oxy)-6-(4-oxypyridine)hexane (BuABO6OPyr) is reported. CB6OBA shows a monotropic twist-bend nematic phase and an enantiotropic nematic phase whereas the two pyridyl-based components do not exhibit liquid crystallinity. Both equimolar mixtures (CB6OBA/BuABOnOPyr) show enantiotropic nematic phases. The nematic-isotropic transition temperature and associated entropy change are higher for the CB6OBA/BuABO6OPyr mixture than for the CB6OBA/BuABO5OPyr mixture. This may be accounted for in terms of the average shapes of the hydrogen-bonded 1:1 complexes formed between the two differing components in the mixtures. However, Fourier transform infrared spectroscopy reveals that this complex is not formed quantitatively, but instead a complex mixture exists over the complete temperature range studied, involving the 1:1 complex, both cyclic and open acid dimers, free acid and hence, free BuABOnOPyr molecules.

Semiconducting Nanotubes by Intrachain Folding Following Macroscopic Assembly of a Naphthalene–Diimide (NDI) Appended Polyurethane

This article describes a well-designed supramolecular assembly of a classical polyurethane scaffold containing pendant n-type semiconducting naphthalene–diimide (NDI) chromophores and consequences on excited state dynamics and charge carrier mobilities. A polycondensation reaction between hexamethylene–diisocyanate and a NDI-containing diol in the presence of a chiral “mono-functional impurity” produced the desired polymer (P1) with a predictable degree of polymerization and end-capping by chiral units. In aliphatic hydrocarbons, such as methylcyclohexane (MCH), P1 adopts a folded conformation with appreciably high thermal stability by intrachain H-bonding among the urethane groups as established by solvent, concentration and temperature-dependent FT-IR and 1H NMR spectroscopy and small angle XRD studies. Folded structure can be further ascertained by the pronounced Cotton effect in MCH owing to the chiral induction by the so-called “sergeant and soldiers” principle from the asymmetric units located only ...

A twist-bend nematic phase driven by hydrogen bonding.

The liquid crystalline phase behavior of 4-[6-(4'-cyanobiphenyl-4-yl)hexyloxy]benzoic acid (CB6OBA) and 4-[5-(4'-cyanobiphenyl-4-yloxy)pentyloxy]benzoic acid (CBO5OBA) is described. Both acids show an enantiotropic nematic phase attributed to the formation of supramolecular complexes by hydrogen bonding between the benzoic acid units. In addition, CB6OBA provides the first example of hydrogen bonding driving the formation of the twist-bend nematic phase. The observation of the twist-bend nematic phase for CB6OBA, but not CBO5OBA, is attributed to the more bent molecular shape of the complexes formed by the former, reinforcing the view that shape is a key factor in stabilizing this new phase. Temperature-dependent FTIR spectroscopy reveals differences in hydrogen bonding between the two nematic phases shown by CB6OBA which suggest that the open hydrogen-bonded complexes may play an important role in stabilizing the helical arrangement found in the twist-bend nematic phase.

Influence of molecular weight on polymorphs and temperature-induced structure evolution of regioregular poly(3-dodecylthiophene)

Formation of form I and form II polymorphs and their phase transitions were investigated for poly(3-dodecylthiophene) (P3DDT) films with molecular weights of 5300, 14 800, and 59 000 respectively, using temperature-dependent FTIR spectroscopy in conjugation with X-ray diffraction and differential scanning calorimetry. For the P3DDTs with the higher molecular weights (14 800 and 59 000), the form I polymorph was obtained by fast drying a solution at ambience or crystallization from the melt, whereas for the low molecular weight one (5300), the form II structure was produced, the amount of which can be increased by slowing down the process. Temperature-induced phase transitions of the three P3DDTs were explored. The results show that form II is thermodynamically more stable at room temperature, but its formation is kinetically hindered, and possible reasons are discussed.

Conformational Heterogeneity and Propagation of Structural Changes in the LOV2/Jα Domain from Avena sativa Phototropin 1 as Recorded by Temperature-Dependent FTIR Spectroscopy

Phototropins control phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Phototropin 1 (phot1) is composed of a kinase domain linked to two blue light-sensing domains, LOV2 and LOV1, which bind flavin mononucleotide. Disruption of the interaction between the LOV2 domain and a helical segment named Jα, joining LOV to the kinase domain, induces the subsequent kinase activity of phototropin 1 and further-downstream signal transduction. Here we study the effects of temperature and hydration on the light-triggered signal propagation in the phot1 LOV2 domain of Avena sativa (AsLOV2/Jα), using Fourier transform infrared spectroscopy to unravel part of the molecular mechanism of phototropin 1. We report that AsLOV2/Jα shows an intense signal in the amide I and II regions, arising mainly from β-sheet changes and the unbinding of the Jα helix from the Per-ARNT-Sim core and its subsequent partial unfolding. Importantly, these structural changes only occur under conditions of full hydration and at temperatures above 280 K. We characterized a newly isolated low-hydration intermediate that shows a downshift of high-frequency amide I signals and that possibly corresponds to loop tightening, without large β-sheet or Jα structural changes. In addition, we report a heterogeneity in AsLOV2/Jα involving two different C(4)=O conformer populations, coexisting in the dark state and characterized by C(4)=O carbonyl frequencies at 1712 cm−1 and 1694 cm−1 that are attributable to a single H-bond and two H-bonds at this site, respectively. Such conformers display slightly shifted absorption spectra and cause a splitting of the 475-nm band in the ultraviolet/visible spectra of LOV domains at low temperature.

A Facile Strategy for the Preparation of Azide Polymers via Room Temperature RAFT Polymerization by Redox Initiation

A new vinyl aryl azide monomer, 4-azidophenyl methacrylate (APM), has been synthesized and characterized by (1) H NMR and FT-IR spectroscopy. The thermal stability of APM has been investigated by temperature-dependent FT-IR spectroscopy and (1) H NMR, and the monomer has been demonstrated to be quite stable at ambient temperature. Reversible addition-fragmentation chain transfer (RAFT) homopolymerization and copolymerizations of APM with methyl acrylate, methyl methacrylate, and styrene have been carried out at room temperature using a redox initiator, benzoyl peroxide (BPO)/N,N-dimethylaniline (DMA). The results show that the polymerizations bear all the characteristics of controlled/living free-radical polymerizations. Moreover, the cycloaddition of azido group to carbon-carbon double bond can be avoided in the polymerization process at room temperature.

Intermolecular Hydrogen Bonds Formed Between Amino Acid Molecules in Aqueous Solution Investigated by Temperature-jump Nanosecond Time-resolved Transient Mid-IR Spectroscopy

Carboxyl (COO) vibrational modes of two amino acids histidine and glycine in D2O solution were investigated by temperature-dependent FTIR spectroscopy and temperature-jump nanosecond time-resolved IR difference absorbance spectroscopy. The results show that hydrogen bonds are formed between amino acid molecules as well as between the amino acid molecule and the solvent molecules. The asymmetric vibrational frequency of COO around 1600-1610 cm1 is blue shifted when raising temperature, indicating that the strength of the hydrogen bonds becomes weaker at higher temperature. Two bleaching peaks at 1604 and 1612 cm1 were observed for histidine in response to a temperature jump from 10 C to 20 C. The lower vibrational frequency at 1604 cm1 is assigned to the chain COO group which forms the intermolecular hydrogen bond with {NH3}+ group, while the higher frequency at 1612 cm1 is assigned to the end COO group forming hydrogen bonds with the solvent molecules. This is because that the hydrogen bonds in the former are expected to be stronger than the latter. In addition the intensities of these two bleaching peaks are almost the same. In contrast, only the lower frequency at 1604 cm1 bleaching peak has been observed for glycine. The fact indicates that histidine molecules form a dimer-like intermolecular chain while glycine forms a relatively longer chain in the solution. The rising phase of the IR absorption kinetics in response to the temperature-jump detected at 1604 cm1 for histidine is about 3010 ns, within the resolution limit of our instrument, indicating that breaking or weakening the hydrogen bond is a very fast process.

Residue Specific Resolution of Protein Folding Dynamics Using Isotope-Edited Infrared Temperature Jump Spectroscopy

A major difficulty in experimental studies of protein folding is the lack of nonperturbing, residue specific probes of folding. Here, we demonstrate the ability to resolve protein folding dynamics at the level of a single residue using 13C=18O isotope-edited infrared spectroscopy. A single 13C=18O isotopic label was incorporated into the backbone of the 36 residue, three-helix bundle villin headpiece subdomain (HP36). The label was placed in a solvent protected region of the second alpha-helix of the protein. The 13C=18O isotopic label shifted the carbonyl stretching frequency to 1572.1 cm-1 in the folded state, well removed from the 12C=16O band of the unlabeled protein backbone. The unique IR signature of the 13C=18O label was exploited to probe the equilibrium thermal unfolding transition using temperature-dependent FTIR spectroscopy. The folding/unfolding dynamics were monitored using temperature-jump (T-jump) IR spectroscopy. The equilibrium unfolding studies showed conformational changes suggestive of a loss of helical structure in helix 2 prior to the global unfolding of the protein. T-jump relaxation kinetics probing both the labeled site and the 12C=16O band were found to be biphasic with similar relaxation rates. The slow relaxation phase (approximately 2 x 10(5) s-1) corresponds to the global folding transition. The location of the label, a buried position in helix 2, provides an important probe of the origin of the fast relaxation phase (approximately 10(7) s-1). This phase has significant amplitude for the labeled position even though it is well protected from solvent in the folded structure. The fast phase likely represents a rapid pre-equilibrium that involves solvent penetration around the label and possible partial unfolding of helix 2 prior to the global unfolding transition. This work represents the first experimental study of ultrafast folding dynamics with residue specific resolution.