Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation.

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca2+-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.

Calcium-induced compaction and clustering of vesicles tracked with molecular resolution.

Theory and simulations predict the complex nature of calcium interaction with the lipid membrane. By maintaining the calcium concentrations at physiological conditions, herein we demonstrate experimentally the effect of Ca2+ in a minimalistic cell-like model. For this purpose, giant unilamellar vesicles (GUVs) with a neutral lipid DOPC are generated, and the ion-lipid interaction is observed with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy providing molecular resolution. Firstly, Ca2+ encapsulated within the vesicle binds to the phosphate head groups of the inner leaflets and triggers vesicle compaction. This is tracked by changes in vibrational modes of the lipid groups. As the calcium concentration within the GUV increases, IR intensities change indicating vesicle dehydration and lateral compression of the membrane. Secondly, by inducing a calcium gradient across the membrane up to a ratio of 1:20, interaction between several vesicles occurs as Ca2+ can bind to the outer leaflets leading to vesicle clustering. It is observed that larger calcium gradients induce stronger interactions. These findings with an exemplary biomimetic model reveal that divalent calcium ions not only cause local changes to the lipid packing but also have macroscopic implications to initiate vesicle-vesicle interaction.

Mid-IR quantum cascade laser spectroscopy to resolve lipid dynamics during the photocycle of bacteriorhodopsin.

Membranes are crucial for the functionality of membrane proteins in several cellular processes. Time-resolved infrared (IR) spectroscopy enables the investigation of interaction-induced dynamics of the protein and the lipid membrane. The photoreceptor and proton pump bacteriorhodopsin (BR) was reconstituted into liposomes, mimicking the native purple membrane. By utilization of deuterated lipid alkyl chains, corresponding vibrational modes are frequency-shifted into a spectrally silent window that allows us to monitor lipid dynamics during the photoreaction of BR. Our home-built quantum cascade laser (QCL)-based IR spectrometer covers all relevant spectral regions to detect both lipid and protein vibrational modes. QCL-probed transients at single wavenumbers are compared with the previously performed step-scan Fourier-transform IR measurements. The absorbance changes of the lipids could be resolved by QCL-measurements with a much better signal-to-noise ratio and with nanosecond time resolution. We found a correlation of the lipid dynamics with the protonation dynamics in the M intermediate. QCL spectroscopy extends the study of the protein's photocycle toward dynamics of the interacting membrane.

Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking.

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

Observation of Oligomeric States Indicates a High Structural Flexibility Required for the Onset of Polyglutamine Fibrillization.

Polyglutamine (polyQ) diseases are caused by misfolding and aggregation of expanded polyQ tracts in the affected protein. PolyQ fibrils have been studied in detail; however, less is known about oligomeric precursor states. By a combination of time-resolved temperature-jump (T-jump) infrared (IR) spectroscopy and an appropriately tailored polyQ model peptide, we succeeded in disentangling conformational dynamics in the heterogeneous ensemble of states evolving during aggregation. Individual structural elements could be differentiated by IR-specific signatures, i.e., hairpin monomers, ß-structured oligomers, and disordered structure. Submillisecond dynamics were observed for early oligomeric states in contrast to the slow dynamics of fibril growth. We propose that a high structural flexibility of oligomers is required to initiate fibril formation, but not after a fibrillar structure has consolidated and the fibril just grows. Our study reveals that structural flexibility changes at different stages in the aggregation process, from fibril initiation to fibril growth.

Solvent-mediated isotope effects strongly influence the early stages of calcium carbonate formation: exploring D2O vs. H2O in a combined computational and experimental approach.

In experimental studies, heavy water (D2O) is employed, e.g., so as to shift the spectroscopic solvent background, but any potential effects of this solvent exchange on reaction pathways are often neglected. While the important role of light water (H2O) during the early stages of calcium carbonate formation has been realized, studies into the actual effects of aqueous solvent exchanges are scarce. Here, we present a combined computational and experimental approach to start to fill this gap. We extended a suitable force field for molecular dynamics (MD) simulations. Experimentally, we utilised advanced titration assays and time-resolved attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. We find distinct effects in various mixtures of the two aqueous solvents, and in pure H2O or D2O. Disagreements between the computational results and experimental data regarding the stabilities of ion associates might be due to the unexplored role of HDO, or an unprobed complex phase behaviour of the solvent mixtures in the simulations. Altogether, however, our data suggest that calcium carbonate formation might proceed "more classically" in D2O. Also, there are indications for the formation of new structures in amorphous and crystalline calcium carbonates. There is huge potential towards further improving the understanding of mineralization mechanisms by studying solvent-mediated isotope effects, also beyond calcium carbonate. Last, it must be appreciated that H2O and D2O have significant, distinct effects on mineralization mechanisms, and that care has to be taken when experimental data from D2O studies are used, e.g., for the development of H2O-based computer models.

PolyQ aggregation studied by model peptides with intrinsic tryptophan fluorophores.

Polyglutamine (polyQ) model peptides are ideally suited to analyze the involvement of glutamines in the disease-related aggregation onset. Here we use a template-assisted design of polyQ-rich hairpin peptides (Trpzip-Qn) to monitor structural stability with fluorescence spectroscopy. The hairpin model imitates the monomeric motif of a polyQ fibril and is stabilized by hydrophobic interactions of two cross-strand pairs of tryptophans (Trps) which are used as fluorophores to report on structural changes. The Trps also frame the polyQ repeats located on each hairpin strand with a different number of glutamines (Qn). Single-stranded sequences mimic the unfolded state and were used as references to differentiate the intrinsic fluorescence signal from the spectral effect caused by structural changes. Temperature-induced hairpin unfolding was monitored by the spectral shift of the Trp fluorescence signal and transition temperatures were determined. The magnitude of the spectral shift indicates the degree of structural disorder. We observed that a longer polyQ repeat is more disordered and weakens the cross-strand Trp-Trp interactions resulting in a decrease of the spectral shift. Aggregation to a fibrillar and more ordered structure shows an increase of the spectral shift. In addition, a band at 280 nm occurs in the spectrum which clearly correlates with the turbidity of the sample and is attributed to scattering of larger aggregated structures. Our study reveals that the number of glutamines, pH and temperature affect structural stability and aggregation of polyQ repeats.

Synuclein Family Members Prevent Membrane Damage by Counteracting α-Synuclein Aggregation.

The 140 amino acid protein α-synuclein (αS) is an intrinsically disordered protein (IDP) with various roles and locations in healthy neurons that plays a key role in Parkinson's disease (PD). Contact with biomembranes can lead to α-helical conformations, but can also act as s seeding event for aggregation and a predominant ß-sheet conformation. In PD patients, αS is found to aggregate in various fibrillary structures, and the shift in aggregation and localization is associated with disease progression. Besides full-length αS, several related polypeptides are present in neurons. The role of many αS-related proteins in the aggregation of αS itself is not fully understood Two of these potential aggregation modifiers are the αS splicing variant αS Δexon3 (Δ3) and the paralog ß-synuclein (ßS). Here, polarized ATR-FTIR spectroscopy was used to study the membrane interaction of these proteins individually and in various combinations. The method allowed a continuous monitoring of both the lipid structure of biomimetic membranes and the aggregation state of αS and related proteins. The use of polarized light also revealed the orientation of secondary structure elements. While αS led to a destruction of the lipid membrane upon membrane-catalyzed aggregation, ßS and Δ3 aggregated significantly less, and they did not harm the membrane. Moreover, the latter proteins reduced the membrane damage triggered by αS. There were no major differences in the membrane interaction for the different synuclein variants. In combination, these observations suggest that the formation of particular protein aggregates is the major driving force for αS-driven membrane damage. The misbalance of αS, ßS, and Δ3 might therefore play a crucial role in neurodegenerative disease.

Template-assisted design of monomeric polyQ models to unravel the unique role of glutamine side chains in disease-related aggregation.

Expanded polyglutamine (polyQ) sequences cause numerous neurodegenerative diseases which are accompanied by the formation of polyQ fibrils. The unique role of glutamines in the aggregation onset is undoubtedly accepted and a lot structural data of the fibrils have been acquired, however side-chain specific structural dynamics inducing oligomerization are not well understood yet. To analyze spectroscopically the nucleation process, we designed various template-assisted glutamine-rich ß-hairpin monomers mimicking the structural motif of a polyQ fibril. In a top-down strategy, we use a template which forms a well-defined stable hairpin in solution, insert polyQ-rich sequences into each strand and monitor the effects of individual glutamines by NMR, CD and IR spectroscopic approaches. The design was further advanced by alternating glutamines with other amino acids (T, W, E, K), thereby enhancing the solubility and increasing the number of cross-strand interacting glutamine side chains. Our spectroscopic studies reveal a decreasing hairpin stability with increased glutamine content and demonstrate the enormous impact of only a few glutamines - far below the disease threshold - to destabilize structure. Furthermore, we could access sub-ms conformational dynamics of monomeric polyQ-rich peptides by laser-excited temperature-jump IR spectroscopy. Both, the increased number of interacting glutamines and higher concentrations are key parameters to induce oligomerization. Concentration-dependent time-resolved IR measurements indicate an additional slower kinetic phase upon oligomer formation. The here presented peptide models enable spectroscopic molecular analyses to distinguish between monomer and oligomer dynamics in the early steps of polyQ fibril formation and in a side-chain specific manner.

Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.

Stable Prenucleation Calcium Carbonate Clusters Define Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) is an intermediate step during the precipitation of calcium carbonate, and is assumed to play a key role in biomineralization processes. Here, we have developed a model where ion association thermodynamics in homogeneous phases determine the liquid-liquid miscibility gap of the aqueous calcium carbonate system, verified experimentally using potentiometric titrations, and kinetic studies based on stopped-flow ATR-FTIR spectroscopy. The proposed mechanism explains the variable solubilities of solid amorphous calcium carbonates, reconciling previously inconsistent literature values. Accounting for liquid-liquid amorphous polymorphism, the model also provides clues to the mechanism of polymorph selection. It is general and should be tested for systems other than calcium carbonate to provide a new perspective on the physical chemistry of LLPS mechanisms based on stable prenucleation clusters rather than un-/metastable fluctuations in biomineralization, and beyond.

Enhanced Sensitivity to Local Dynamics in Peptides by Use of Temperature-Jump IR Spectroscopy and Isotope Labeling.

Site-specific isotopic labeling of molecules is a widely used approach in IR spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, as well as on hydrogen bonding and vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded ß-sheet peptide and by use of selected 13 C isotope substitutions at multiple positions in the peptide backbone. Single-strand labels give rise to isotopically shifted bands at different frequencies, depending on the specific sites; this demonstrates sensitivity to the local environment. Cross-strand double- and triple-labeled peptides exhibited two resolved bands that could be uniquely assigned to specific residues, the equilibrium IR spectra of which indicated only weak local-mode coupling. Temperature-jump IR laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them, relative to that of equilibrium FTIR spectroscopy. Site-specific relaxation rates were altered upon the introduction of additional cross-strand isotopes. Likewise, the rates for the global ß-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. This study reveals that isotope labels provide not only local structural probes, but rather sense the dynamic complexity of the molecular environment.

Immobilization approaches can affect protein dynamics: a surface-enhanced infrared spectroscopic study on lipid-protein interactions.

The intrinsically disordered Parkinson disease protein α-synuclein (αS) performs conformational changes induced by intermolecular protein-protein as well as by protein-membrane interactions. Aggregation of αS is a hallmark for the disease, however the role of the membrane in the aggregation process still needs to be clarified. We used a surface-enhanced infrared absorption (SEIRA) spectroscopic approach to investigate the effect of lipid interactions on αS conformation. The near-field detection of SEIRA allows to study exclusively structural changes of immobilized αS with the advantage that the supernatant remains undetected and thus does not interfere with the spectral read-out. self-assembled monolayer (SAMs) of mixed NHS-PEG-SH linker and MT(PEG)4 spacer molecules were utilized to immobilize αS. The linker/spacer composition of the SAM was adjusted to prevent αS-αS interactions. Two different methods were applied for site-specific (C-terminal and N-terminal) αS immobilization. The immobilized protein was then exposed to lipid vesicles and SEIRA difference spectra were recorded to monitor the αS conformation over time. Irrespective of the used immobilization method, αS tethering hindered lipid-induced conformational changes. The spectra also indicate that a fraction of the immobilized αS eventually desorbs from the surface into the supernatant solution. Desorbed αS performs conformational changes and formation of ß-structured aggregates is observed upon interaction with either lipid vesicles or supplementary αS. Our study demonstrates that αS aggregates only when the protein is free in solution and that surface immobilization procedures, commonly used in many analytical applications, can change the dynamic behavior of proteins thereby affecting protein structure and function.

Interactions of p53 with poly(ADP-ribose) and DNA induce distinct changes in protein structure as revealed by ATR-FTIR spectroscopy.

Due to multiple domains and in part intrinsically disordered regions, structural analyses of p53 remain a challenging task, particularly in complex with DNA and other macromolecules. Here, we applied a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic approach to investigate changes in secondary structure of full-length p53 induced by non-covalent interactions with DNA and poly(ADP-ribose) (PAR). To validate our approach, we confirmed a positive regulatory function of p53's C-terminal domain (CTD) with regard to sequence-specific DNA binding and verified that the CTD mediates p53-PAR interaction. Further, we demonstrate that DNA and PAR interactions result in distinct structural changes of p53, indicating specific binding mechanisms via different domains. A time-dependent analysis of the interplay of DNA and PAR binding to p53 revealed that PAR represents p53's preferred binding partner, which efficiently controls p53-DNA interaction. Moreover, we provide infrared spectroscopic data on PAR pointing to the absence of regular secondary structural elements. Finally, temperature-induced melting experiments via CD spectroscopy show that DNA binding stabilizes the structure of p53, while PAR binding can shift the irreversible formation of insoluble p53 aggregates to higher temperatures. In conclusion, this study provides detailed insights into the dynamic interplay of p53 binding to DNA and PAR at a formerly inaccessible molecular level.

Isotopically Site-Selected Dynamics of a Three-Stranded ß-Sheet Peptide Detected with Temperature-Jump Infrared-Spectroscopy.

Infrared detected temperature-jump (T-jump) spectroscopy and site-specific isotopic labeling were applied to study a model three-stranded ß-sheet peptide with the goal of individually probing the dynamics of strand and turn structural elements. This peptide had two DPro-Gly (pG) turn sequences to stabilize the two component hairpins, which were labeled with 13C═O on each of the Gly residues to resolve them spectroscopically. Labeling the second turn on the amide preceding the DPro (Xxx-DPro amide) provided an alternate turn label as a control. Placing 13C═O labels on specific in-strand residues gave shifted modes that overlap the Xxx-DPro amide I' modes. Their impact could be separated from the turn dynamics by a novel difference transient analysis approach. Fourier-transform infrared spectra were modeled with density functional theory-computations which showed the local, isotope-selected vibrations were effectively uncoupled from the other amide I modes. Our T-jump dynamics results, combined with nuclear magnetic resonance structures and equilibrium spectral measurements, showed the first turn to be most stable and best formed with the slowest dynamics, whereas the second turn and first strand (N-terminus) had similar dynamics, and the third strand (C-terminus) had the fastest dynamics and was the least structured. The relative dynamics of the strands, Xxx-DPro amides, and 13C-labeled Gly residues on the turns also qualitatively corresponded to molecular dynamics (MD) simulations of turn and strand fluctuations. MD trajectories indicated the turns to be bistable, with the first turn being Type I' and the second turn flipping from I' to II'. The differences in relaxation times for each turn and the separate strands revealed that the folding process of this turn-stabilized ß-sheet structure proceeds in a multistep process.

Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths.

Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish ß-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a ß-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various ß-structured intermediates, including intramolecular ß-sheets.

The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.

Role of Aromatic Cross-Links in Structure and Dynamics of Model Three-Stranded ß-Sheet Peptides.

A series of closely related peptide sequences that form triple-strand structures was designed with a variation of cross-strand aromatic interactions and spectroscopically studied as models for ß-sheet formation and stabilities. Structures of the three-strand models were determined with NMR methods and temperature-dependent equilibrium studies performed using circular dichroism and Fourier transform infrared spectroscopies. Our equilibrium data show that the presence of a direct cross-strand aromatic contact in an otherwise folded peptide does not automatically result in an increased thermal stability and can even distort the structure. The effect on the conformational dynamics was studied with infrared-detected temperature-jump relaxation methods and revealed a high sensitivity to the presence and the location of the aromatic cross-links. Aromatic contacts in the three-stranded peptides slow down the dynamics in a site-specific manner, and the impact seems to be related to the distance from the turn. With a Xxx-DPro linkage as a probe with some sensitivity for the turn, small differences were revealed in the relative relaxation of the sheet strands and turn regions. In addition, we analyzed the component hairpins, which showed less uniform dynamics as compared to the parent three-stranded ß-sheet peptides.

Simultaneous IR-Spectroscopic Observation of α-Synuclein, Lipids, and Solvent Reveals an Alternative Membrane-Induced Oligomerization Pathway.

The intrinsically disordered protein α-synuclein (αS), a known pathogenic factor for Parkinson's disease, can adopt defined secondary structures when interacting with membranes or during fibrillation. The αS-lipid interaction and the implications of this process for aggregation and damage to membranes are still poorly understood. Therefore, we established a label-free infrared (IR) spectroscopic approach to allow simultaneous monitoring of αS conformation and membrane integrity. IR showed its unique sensitivity for identifying distinct ß-structured aggregates. A comparative study of wild-type αS and the naturally occurring splicing variant αS Δexon3 yielded new insights into the membrane's capability for altering aggregation pathways.

Biomolecular dynamics studied with IR-spectroscopy using quantum cascade lasers combined with nanosecond perturbation techniques.

Early events of protein folding can be studied with fast perturbation techniques triggering non-equilibrium relaxation dynamics. A nanosecond laser-excited pH-jump or temperature-jump (T-jump) was applied to initiate helix folding or unfolding of poly-l-glutamic acid (PGA). PGA is a homopolypeptide with titratable carboxyl side-chains whose protonation degree determines the PGA conformation. A pH-jump was realized by the photochemical release of protons and induces PGA folding due to protonation of the side-chains. Otherwise, the helical conformation can be unfolded by a T-jump. We operated under conditions where PGA does not aggregate and temperature and pH are the regulatory properties of its conformation. The experiments were performed in such a manner that the folding/unfolding jump proceeded to the same PGA conformation. We quantified the increase/decrease in helicity induced by the pH-/T-jump and demonstrated that the T-jump results in a relatively small change in helical content in contrast to the pH-jump. This is caused by the strong pH-dependence of the PGA conformation. The conformational changes were detected by time-resolved single wavelength IR-spectroscopy using quantum cascade lasers (QCL). We could independently observe the kinetics for α-helix folding and unfolding in PGA by using different perturbation techniques and demonstrate the high sensitivity of time-resolved IR-spectroscopy to study protein folding mechanisms.