HYPK: A marginally disordered protein sensitive to charge decoration.

Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.

A switch from α-helical to ß-strand conformation during co-translational protein folding.

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a ß-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a ß-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity.

Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy.

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.

Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2 dimer probes of local DNA conformations at or near functional protein binding sites.

Exciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of 'internally-labeled' (iCy3)2 dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2 dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2 dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.

Comprehensive analysis of intramolecular G-quadruplex structures: furthering the understanding of their formalism.

G-quadruplexes (G4) are helical structures found in guanine-rich DNA or RNA sequences. Generally, their formalism is based on a few dozen structures, which can produce some inconsistencies or incompleteness. Using the website ASC-G4, we analyzed the structures of 333 intramolecular G4s, of all types, which allowed us to clarify some key concepts and present new information. To each of the eight distinguishable topologies corresponds a groove-width signature and a predominant glycosidic configuration (gc) pattern governed by the directions of the strands. The relative orientations of the stacking guanines within the strands, which we quantified and related to their vertical gc successions, determine the twist and tilt of the helices. The latter impact the minimum groove widths, which represent the space available for lateral ligand binding. The G4 four helices have similar twists, even when these twists are irregular, meaning that they have various angles along the strands. Despite its importance, the vertical gc succession has no strict one-to-one relationship with the topology, which explains the discrepancy between some topologies and their corresponding circular dichroism spectra. This study allowed us to introduce the new concept of platypus G4s, which are structures with properties corresponding to several topologies.

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae.

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread.

G-quadruplex (G4) formed by repetitive guanosine-rich sequences plays important roles in diverse cellular processes; however, its roles in viral infection are not fully understood. In this study, we investigated the genome-wide distribution of G4-forming sequences (G4 motifs) in Varicella-Zoster virus (VZV) and found that G4 motifs are enriched in the internal repeat short and the terminal repeat short regions flanking the unique short region and also in some reiteration (R) sequence regions. A high density of G4 motifs in the R2 region was found on the template strand of ORF14, which encodes glycoprotein C (gC), a virulent factor for viral growth in skin. Analyses such as circular dichroism spectroscopy, thermal difference spectra, and native polyacrylamide gel electrophoresis with oligodeoxynucleotides demonstrated that several G4 motifs in ORF14 form stable G4 structures. In transfection assays, gC expression from the G4-disrupted ORF14 gene was increased at the transcriptional level and became more resistant to suppression by G4-ligand treatment. The recombinant virus containing the G4-disrupted ORF14 gene expressed a higher level of gC mRNA, while it showed a slightly reduced growth. This G4-disrupted ORF14 virus produced smaller plaques than the wild-type virus. Our results demonstrate that G4 formation via reiteration sequences suppresses gC expression during VZV infection and regulates viral cell-to-cell spread.

Essential role of coiled coils for aggregation and activity of Q/N-rich prions and PolyQ proteins.

The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming ß sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.

NACDDB: Nucleic Acid Circular Dichroism Database.

The Nucleic Acid Circular Dichroism Database (NACDDB) is a public repository that archives and freely distributes circular dichroism (CD) and synchrotron radiation CD (SRCD) spectral data about nucleic acids, and the associated experimental metadata, structural models, and links to literature. NACDDB covers CD data for various nucleic acid molecules, including DNA, RNA, DNA/RNA hybrids, and various nucleic acid derivatives. The entries are linked to primary sequence and experimental structural data, as well as to the literature. Additionally, for all entries, 3D structure models are provided. All entries undergo expert validation and curation procedures to ensure completeness, consistency, and quality of the data included. The NACDDB is open for submission of the CD data for nucleic acids. NACDDB is available at: https://genesilico.pl/nacddb/.

Inhibited complete folding of consecutive human telomeric G-quadruplexes.

Noncanonical DNA structures, termed G-quadruplexes, are present in human genomic DNA and are important elements in many DNA metabolic processes. Multiple sites in the human genome have G-rich DNA stretches able to support formation of several consecutive G-quadruplexes. One of those sites is the telomeric overhang region that has multiple repeats of TTAGGG and is tightly associated with both cancer and aging. We investigated the folding of consecutive G-quadruplexes in both potassium- and sodium-containing solutions using single-molecule FRET spectroscopy, circular dichroism, thermal melting and molecular dynamics simulations. Our observations show coexistence of partially and fully folded DNA, the latter consisting of consecutive G-quadruplexes. Following the folding process over hours in sodium-containing buffers revealed fast G-quadruplex folding but slow establishment of thermodynamic equilibrium. We find that full consecutive G-quadruplex formation is inhibited by the many DNA structures randomly nucleating on the DNA, some of which are off-path conformations that need to unfold to allow full folding. Our study allows describing consecutive G-quadruplex formation in both nonequilibrium and equilibrium conditions by a unified picture, where, due to the many possible DNA conformations, full folding with consecutive G-quadruplexes as beads on a string is not necessarily achieved.

Steric hindrance and structural flexibility shape the functional properties of a guanine-rich oligonucleotide.

Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers.

Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation.

Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet, some protein folds are easier to design than others. Previous work identified the 43-residue ɑßßɑ fold as especially challenging: The best designs had only a 2% success rate, compared to 39 to 87% success for other simple folds [G. J. Rocklin et al., Science 357, 168-175 (2017)]. This suggested the ɑßßɑ fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over 10,000 new ɑßßɑ proteins and found over 3,000 of them to fold into stable structures using a high-throughput protease-based assay. NMR, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed ɑßßɑ structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the ɑßßɑ topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

Efficient enumeration and visualization of helix-coil ensembles.

Helix-coil models are routinely used to interpret circular dichroism data of helical peptides or predict the helicity of naturally-occurring and designed polypeptides. However, a helix-coil model contains significantly more information than mean helicity alone, as it defines the entire ensemble-the equilibrium population of every possible helix-coil configuration-for a given sequence. Many desirable quantities of this ensemble are either not obtained as ensemble averages or are not available using standard helicity-averaging calculations. Enumeration of the entire ensemble can allow calculation of a wider set of ensemble properties, but the exponential size of the configuration space typically renders this intractable. We present an algorithm that efficiently approximates the helix-coil ensemble to arbitrary accuracy by sequentially generating a list of the M highest populated configurations in descending order of population. Truncating this list of (configuration, population) pairs at a desired accuracy provides an approximating sub-ensemble. We demonstrate several uses of this approach for providing insight into helix-coil ensembles and folding mechanisms, including landscape visualization.

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors.

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Lessons on protein structure from interleukin-4: All disulfides are not created equal.

Interleukin-4 (IL-4) is a hematopoietic cytokine composed by a four-helix bundle stabilized by an antiparallel beta-sheet and three disulfide bonds: Cys3-Cys127, Cys24-Cys65, and Cys46-Cys99. IL-4 is involved in several immune responses associated to infection, allergy, autoimmunity, and cancer. Besides its physiological relevance, IL-4 is often used as a "model" for protein design and engineering. Hence, to understand the role of each disulfide in the structure and dynamics of IL-4, we carried out several spectroscopic analyses (circular dichroism [CD], fluorescence, nuclear magnetic resonance [NMR]), and molecular dynamics (MD) simulations on wild-type IL-4 and four IL-4 disulfide mutants. All disulfide mutants showed loss of structure, altered interhelical angles, and looser core packings, showing that all disulfides are relevant for maintaining the overall fold and stability of the four-helix bundle motif, even at very low pH. In the absence of the disulfide connecting both protein termini Cys3-Cys127, C3T-IL4 showed a less packed protein core, loss of secondary structure (~9%) and fast motions on the sub-nanosecond time scale (lower S2 order parameters and larger τc correlation time), especially at the two protein termini, loops, beginning of helix A and end of helix D. In the absence of Cys24-Cys65, C24T-IL4 presented shorter alpha-helices (14% loss in helical content), altered interhelical angles, less propensity to form the small anti-parallel beta-sheet and increased dynamics. Simultaneously deprived of two disulfides (Cys3-Cys127 and Cys24-Cys65), IL-4 formed a partially folded "molten globule" with high 8-anilino-1-naphtalenesulphonic acid-binding affinity and considerable loss of secondary structure (~50%decrease), as shown by the far UV-CD, NMR, and MD data.

An Atomic and Molecular Insight into How PFOA Reduces α-Helicity, Compromises Substrate Binding, and Creates Binding Pockets in a Model Globular Protein.

Per- and polyfluoroalkyl substances (PFAS) pose significant health risks due to their widespread presence in various environmental and biological matrices. However, the molecular-level mechanisms underlying the interactions between PFAS and biological constituents, including proteins, carbohydrates, lipids, and DNA, remain poorly understood. Here, we investigate the interactions between a legacy PFAS, viz. perfluorooctanoic acid (PFOA), and the milk protein ß-lactoglobulin (BLG) obtained using a combination of experimental and computational techniques. Circular dichroism studies reveal that PFOA perturbs the secondary structure of BLG, by driving a dose-dependent loss of α-helicity and alterations in its ß-sheet content. Furthermore, exposure of the protein to PFOA attenuates the on-rate constant for the binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANS), suggesting potential functional impairment of BLG by PFOA. Steered molecular dynamics and umbrella sampling calculations reveal that PFOA binding leads to the formation of an energetically favorable novel binding pocket within the protein, when residues 129-142 are steered to unfold from their initial α-helical structure, wherein a host of intermolecular interactions between PFOA and BLG's residues serve to insert the PFOA into the region between the unfolded helix and beta-sheets. Together, the data provide a novel understanding of the atomic and molecular mechanism(s) by which PFAS modulates structure and function in a globular protein, leading to a beginning of our understanding of altered biological outcomes.

A metabolite binding protein moonlights as a bile-responsive chaperone.

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.

γ-Secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics.

Sequence variants of the microglial expressed TREM2 (triggering receptor expressed on myeloid cells 2) are a major risk factor for late onset Alzheimer's disease. TREM2 requires a stable interaction with DAP12 in the membrane to initiate signaling, which is terminated by TREM2 ectodomain shedding and subsequent intramembrane cleavage by γ-secretase. To understand the structural basis for the specificity of the intramembrane cleavage event, we determined the solution structure of the TREM2 transmembrane helix (TMH). Caused by the presence of a charged amino acid in the membrane region, the TREM2-TMH adopts a kinked structure with increased flexibility. Charge removal leads to TMH stabilization and reduced dynamics, similar to its structure in complex with DAP12. Strikingly, these dynamical features match with the site of the initial γ-secretase cleavage event. These data suggest an unprecedented cleavage mechanism by γ-secretase where flexible TMH regions act as key determinants of substrate cleavage specificity.

Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure.

Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose-response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.

Dynamic Observation of the Membrane Interaction Processes of ß-Lactoglobulin by Time-Resolved Vacuum-Ultraviolet Circular Dichroism.

The elucidation of protein-membrane interactions is pivotal for comprehending the mechanisms underlying diverse biological phenomena and membrane-related diseases. In this investigation, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy, utilizing synchrotron radiation (SR), was employed to dynamically observe membrane interaction processes involving water-soluble proteins at the secondary-structure level. The study utilized a time-resolved (TR) T-shaped microfluidic cell, facilitating the rapid and efficient mixing of protein and membrane solutions. This system was instrumental in acquiring measurements of the time-resolved circular dichroism (TRCD) spectra of ß-lactoglobulin (bLG) during its interaction with lysoDMPG micelles. The results indicate that bLG undergoes a ß-α conformation change, leading to the formation of the membrane-interacting state (M-state), with structural alterations occurring in more than two steps. Global fitting analysis, employing biexponential functions with all of the TRCD spectral data sets, yielded two distinct rate constants (0.18 ± 0.01 and 0.06 ± 0.003/s) and revealed a unique spectrum corresponding to an intermediate state (I-state). Secondary-structure analysis of bLG in its native (N-, I-, and M-states) highlighted that structural changes from the N- to I-states predominantly occurred in the N- and C-terminal regions, which were prominently exposed to the membrane. Meanwhile, transitions from the I- to M-states extended into the inner barrel regions of bLG. Further examination of the physical properties of α-helical segments, such as effective charge and hydrophobicity, revealed that the N- to I- and I- to M-state transitions, which are ascribed to first- and second-rate constants, respectively, are primarily driven by electrostatic and hydrophobic interactions, respectively. These findings underscore the capability of the TR-VUVCD system as a robust tool for characterizing protein-membrane interactions at the molecular level.