Unfolded Chains Research Articles

Loop formation and translational diffusion of intrinsically disordered proteins.

The conformational flexibility and dynamics of unfolded peptide chains is of major interest in the context of protein folding and protein functioning. The rate with which amino acids at different positions along the peptide chain meet sets an upper speed limit for protein folding. By using single-molecule photo-induced energy transfer spectroscopy, we have systematically measured end-to-end and end-to-internal site contact formation rates for several intrinsically disordered protein fragments (IDPs) (11 to 41 amino acids) and have also determined their hydrodynamic radius using dual-focus fluorescence correlation spectroscopy. For interpreting the measured values, we have developed a Brownian dynamics model (based on bead-rod chain dynamics in a thermal bath including hydrodynamic interactions) which quantitatively reproduces all measured data surprisingly well while requiring only two fit parameters. The model provides a complete picture of the peptides' dynamics and allows us to translate the experimental rates and radii into molecular properties of the peptides: We find a persistence length of l_{P}=5.2±1.9Å, a hydrodynamic radius of a=3.5±0.7Å per amino acid, and that excluded volume effects play an important role in the dynamics of IDPs.

The Anti-Aggregation Holdase Hsp33 Promotes the Formation of Folded Protein Structures

Holdase chaperones are known to be central to suppressing aggregation, but how they affect substrate conformations remains poorly understood. Here, we use optical tweezers to study how the holdase Hsp33 alters folding transitions within single maltose binding proteins and aggregation transitions between maltose binding protein substrates. Surprisingly, we find that Hsp33 not only suppresses aggregation but also guides the folding process. Two modes of action underlie these effects. First, Hsp33 binds unfolded chains, which suppresses aggregation between substrates and folding transitions within substrates. Second, Hsp33 binding promotes substrate states in which most of the chain is folded and modifies their structure, possibly by intercalating its intrinsically disordered regions. A statistical ensemble model shows how Hsp33 function results from the competition between these two contrasting effects. Our findings reveal an unexpectedly comprehensive functional repertoire for Hsp33 that may be more prevalent among holdases and dispels the notion of a strict chaperone hierarchy.

NMR characterization of solvent accessibility and transient structure in intrinsically disordered proteins

In order to understand the conformational behavior of intrinsically disordered proteins (IDPs) and their biological interaction networks, the detection of residual structure and long-range interactions is required. However, the large number of degrees of conformational freedom of disordered proteins require the integration of extensive sets of experimental data, which are difficult to obtain. Here, we provide a straightforward approach for the detection of residual structure and long-range interactions in IDPs under near-native conditions using solvent paramagnetic relaxation enhancement (sPRE). Our data indicate that for the general case of an unfolded chain, with a local flexibility described by the overwhelming majority of available combinations, sPREs of non-exchangeable protons can be accurately predicted through an ensemble-based fragment approach. We show for the disordered protein α-synuclein and disordered regions of the proteins FOXO4 and p53 that deviation from random coil behavior can be interpreted in terms of intrinsic propensity to populate local structure in interaction sites of these proteins and to adopt transient long-range structure. The presented modification-free approach promises to be applicable to study conformational dynamics of IDPs and other dynamic biomolecules in an integrative approach.

Mechanochemistry in Translation.

As the influence of translation rates on protein folding and function has come to light, the mechanisms by which translation speed is modulated have become an important issue. One mechanism entails the generation of force by the nascent protein. Cotranslational processes, such as nascent protein folding, the emergence of unfolded nascent chain segments from the ribosome's exit tunnel, and insertion of the nascent chain into or translocation of the nascent chain through membranes, can generate forces that are transmitted back to the peptidyl transferase center and affect translation rates. In this Perspective, we examine the processes that generate these forces, the mechanisms of transmission along the ribosomal exit tunnel to the peptidyl transferase center, and the effects of force on the ribosome's catalytic cycle. We also discuss the physical models that have been developed to predict and explain force generation for individual processes and speculate about other processes that may generate forces that have yet to be tested.

Effects of an in situ instructional design based postpartum hemorrhage simulation training on patient outcomes: an uncontrolled before-and-after study

Objective: To compare postpartum hemorrhage (PPH) patient outcomes before and after an in situ instructional design based PPH simulation attended by obstetrics and gynecology (OBGYN) residents.Methods: This uncontrolled before-and-after study was conducted in Recife, Brazil including all 1388 women delivering from June to August 2012 and all 1357 delivering from June to August 2013. The 36 OBGYN residents were divided into13 teams of two or three participants and were trained through ID based PPH simulation training with the following eight steps: (1) prior knowledge activation, (2) video demonstration, (3) dual-coding PPH protocol discussion—an image association during the training, (4) training scenario # 1, (5) debriefing, (6) training scenario # 2 with immediate feedback, (7) training scenario # 3, and (8) debriefing with self-assessment. The training scenarios had an increasing level of complexity. The main goal of the training was the adequate management of PPH and situational awareness improvement—the ability to anticipate, recognize, and intercept unfolding error chains. The primary patient outcomes rates used for the before and after comparison were therapeutic uterotonics use within 24 h of birth and blood transfusion. Secondary outcomes were therapeutic oxytocin mean dosage IU within 24 h of birth, postpartum Hb < 6 g/dL, among others. Chi-square test was used for categorical variables comparison and independent t-test for continuous variables.Results: PPH rates were 100 (7.2% of 2012 deliveries) and 80 cases (5.9% of 2013 deliveries), respectively. Comparison of primary post- and pre-simulation outcomes revealed no significant differences. However, in the comparison for therapeutic oxytocin mean dosage IU within 24 h of birth, there was an increase found after the simulation (15.98 ± 7.4 versus 25.1 ± 12.3; p < .001). For all other outcome measures, there were no statistical differences.Conclusions: In situ ID based PPH simulation leads to an increase in the mean dosage of oxytocin after training, in selected cases. This may indicate better situational awareness when managing women with PPH.

Free and Chaperone Bound Unfolded States of Outer Membrane Proteins

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with chaperones. In the case of bacterial outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because they remain unfolded for extended periods of time during their biogenesis and rely on interactions with periplasmic chaperones to prevent aggregation and support correct folding. Here, we study the unfolded-state properties of outer-membrane proteins. Using a combination of ensemble and single-molecule spectroscopy techniques including single-molecule FRET, we find that under strongly denaturing conditions and in the absence of chaperones, OMPs adopt conformationally heterogeneous unfolded states that lack the fast chain reconfiguration motions expected for an unstructured, fully unfolded chain. Interestingly, when complexed with periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA), we observe for the protein OmpLA adopts an expanded, yet squished conformational ensemble, in which the overall broad distribution of unfolded states is preserved. Differently sized OMPs like OmpX (8 β-strands) and OmpF (16 β-strands) allow a broader perspective on chaperone enabled conformations of OMPs. These findings indicate that periplasmic chaperones Skp and SurA prestructure OMPs for their next binding partner (e.g., the β-barrel assembly machinery (BAM)) to facilitate insertion and proper folding into the outer membrane.

Theoretical Saxs Signatures of Conformational Heterogeneity and Homogeneity of Disordered Protein Ensembles

The biological function of an intrinsically disordered protein (IDP) is based upon its conformational ensemble which is in turn governed by the IDP's amino acid sequence. In view of the different functions served by different IDPs, this ensemble-function view reinforces the physical expectation that the disordered chains of non-homopolymeric amino acid sequences---IDPs and unfolded chains of globular proteins---are heterogeneous, i.e., their ensemble distributions of conformations are different from those of homopolymers that habor a uniform intrachain interaction. Indeed, recent analyses indicate that conformational heterogeneity is key to reconciling the apparent inconsistency between IDP dimensions determined by SAXS and FRET. This development notwithstanding, it was stated recently that SAXS data on disordered proteins can be adequately accounted for by homopolymer chain models. It is important, therefore, to address the information content of SAXS data, to assess to what extent SAXS data can discriminate between heterogeneous and homogeneous conformational ensembles. We have conducted extensive simulations of subensembles of chain conformations with narrow ranges of radius of gyration Rg and end-to-end distance REE, and compared the molecular form factors (MFFs) and dimensionless Kratky plots of these highly heterogeneous (sub)ensembles with those computed for homogeneous ensembles. We find that the MFFs of heterogeneous and homogeneous ensembles are quite similar when the chain conformations are relatively open, suggesting that SAXS may not be capable of clearly distinguishing heterogeneous from homogeneous conformational ensembles of well-solvated or slightly compact chains. In contrast, MFFs for more compact ensembles are more sensitive to conformational heterogeneity. We have also developed an analytical theory for sequence-dependent MFFs. The theory provides insight into how different conformational ensembles that share the same average REE can have different average Rg's. Ramifications of our findings for the interpretation of experimental data are discussed.

Hyperbolic Pressure-Temperature Phase Diagram of the Zinc-Finger Protein apoKti11 Detected by NMR Spectroscopy.

For a comprehensive understanding of the thermodynamic state functions describing the stability of a protein, the influence of the intensive properties of temperature and pressure has to be known. With the zinc-finger-containing Kti11, we found a suitable protein for this purpose because folding and unfolding transitions occur at an experimentally accessible temperature (280-330 °K) and pressure (0.1-240 MPa) range. We solved the crystal structure of the apo form of Kti11 to reveal two disulfide bonds at the metal-binding site, which seals off a cavity in the β-barrel part of the protein. From a generally applicable proton NMR approach, we could determine the populations of folded and unfolded chains under all conditions, leading to a hyperbolic pressure-temperature phase diagram rarely observed for proteins. A global fit of a two-state model to all derived populations disclosed reliable values for the change in Gibbs free energy, volume, entropy, heat capacity, compressibility, and thermal expansion upon unfolding. The unfolded state of apoKti11 has a lower compressibility compared to the native state and a smaller volume at ambient pressure. Therefore, a pressure increase up to 200 MPa reduces the population of the native state, and above this value, the native population increases again. Pressure-induced chemical-shift changes in two-dimensional 1H-15N NMR spectra could be employed for a molecular interpretation of the thermodynamic properties of apoKti11.

Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide

Phosphoproteomics studies have reported phosphorylation at multiple sites within collagen, raising the possibility that these post-translational modifications regulate the physical or biological properties of collagen. In this study, molecular dynamics simulations and experimental studies were carried out on model peptides to establish foundational principles of phosphorylation of Ser residues in collagen. A (Gly-Xaa-Yaa)11 peptide was designed to include a Ser-containing sequence from type I collagen that was reported to be phosphorylated. The physiological kinase involved in collagen phosphorylation is not known. In vitro studies showed that a model kinase ERK1 (extracellular signal-regulated protein kinase 1) would phosphorylate Ser within the consensus sequence if the collagen-like peptide is in the denatured state but not in the triple-helical state. The peptide was not a substrate for FAM20C, a kinase present in the secretory pathway, which has been shown to phosphorylate many extracellular matrix proteins. The unfolded single chain (Gly-Xaa-Yaa)11 peptide containing phosphoSer was able to refold to form a stable triple helix but at a reduced folding rate and with a small decrease in thermal stability relative to the nonphosphorylated peptide at neutral pH. These biophysical studies on model peptides provide a basis for investigations into the physiological consequences of collagen phosphorylation and the application of phosphorylation to regulate the properties of collagen biomaterials.

Visualizing collagen proteolysis by peptide hybridization: From 3D cell culture to in vivo imaging

Degradation of the extracellular matrix (ECM) is one of the fundamental factors contributing to a variety of life-threatening or disabling pathological conditions. However, a thorough understanding of the degradation mechanism and development of new ECM-targeting diagnostics are severely hindered by a lack of technologies for direct interrogation of the ECM structures at the molecular level. Previously we demonstrated that the collagen hybridizing peptide [CHP, sequence: (GPO)9, O: hydroxyproline] can specifically recognize the degraded and unfolded collagen chains through triple helix formation. Here we show that fluorescently labeled CHP robustly visualizes the pericellular matrix turnover caused by proteolytic migration of cancer cells within 3D collagen culture, without the use of synthetic fluorogenic matrices or genetically modified cells. To facilitate in vivo imaging, we modified the CHP sequence by replacing each proline with a (2S,4S)-4-fluoroproline (f) residue which interferes with the peptide's inherent propensity to self-assemble into homo-triple helices. We show that the new CHP, (GfO)9, tagged with a near-infrared fluorophore, enables in vivo imaging and semi-quantitative assessment of osteolytic bone lesions in mouse models of multiple myeloma. Compared to conventional techniques (e.g., micro-CT), CHP-based imaging is simple and versatile in vitro and in vivo. Therefore, we envision CHP's applications in broad biomedical contexts ranging from studies of ECM biology and drug efficiency to development of clinical molecular imaging.

NMR analyzes protein as it folds

Researchers have long wanted to better understand the way an unfolded protein chain reaches its final folded structure. But experimental tools needed to probe this complex process have been limited. An apparatus developed by Philip Anfinrud, Ad Bax, and coworkers at the National Institutes of Health now opens a new window on the process by allowing nuclear magnetic resonance spectroscopy to follow protein folding at an atomic level of detail in real time (Proc. Natl. Acad. Sci. USA 2018, DOI: 10.1073/pnas.1803642115). Proteins unfold when subjected to high pressure and then refold when normal pressure is restored, a handy trick that researchers pair with spectroscopic techniques to track folding. Fluorescence spectroscopy has been used in this way, but it provides only low-resolution information. NMR can obtain highly precise data at every atom position that naturally contains or can be endowed with a magnetic isotope, like hydrogen-1, carbon-13, or nitrogen-15. But NMR

Chemical stresses fail to mimic the unfolded protein response resulting from luminal load with unfolded polypeptides

The stress sensors ATF6, IRE1, and PERK monitor deviations from homeostatic conditions in the endoplasmic reticulum (ER), a protein biogenesis compartment of eukaryotic cells. Their activation elicits unfolded protein responses (UPR) to re-establish proteostasis. UPR have been extensively investigated in cells exposed to chemicals that activate ER stress sensors by perturbing calcium, N-glycans, or redox homeostasis. Cell responses to variations in luminal load with unfolded proteins are, in contrast, poorly characterized. Here, we compared gene and protein expression profiles in HEK293 cells challenged with ER stress-inducing drugs or expressing model polypeptides. Drug titration to limit up-regulation of the endogenous ER stress reporters heat shock protein family A (Hsp70) member 5 (BiP/HSPA5) and homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERP/HERPUD1) to levels comparable with luminal accumulation of unfolded proteins substantially reduced the amplitude of both transcriptional and translational responses. However, these drug-induced changes remained pleiotropic and failed to recapitulate responses to ER load with unfolded proteins. These required unfolded protein association with BiP and induced a much smaller subset of genes participating in a chaperone complex that binds unfolded peptide chains. In conclusion, UPR resulting from ER load with unfolded proteins proceed via a well-defined and fine-tuned pathway, whereas even mild chemical stresses caused by compounds often used to stimulate UPR induce cellular responses largely unrelated to the UPR or ER-mediated protein secretion.

Non‐Native Structure Present in the Unfolded Ensemble May Initiate Aggregation of ALS variants of Superoxide Dismutase (SOD1)

Aggregates of Cu, Zn Superoxide Dismutase (SOD1) in the spinal cord are a hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Mutations throughout the SOD1 sequence have been found to cause ALS, suggesting a general gain‐of‐function mechanism for the toxicity of this disease. We have previously argued that the aggregation of the unfolded chain, following synthesis on the ribosome and prior to maturation, provides a parsimonious explanation for the common phenotype. To determine whether segments of unfolded SOD1 are capable of adopting structure prior to the exceedingly slow (&gt;10 sec) productive folding reaction, we dissected SOD1 into peptides that were labeled with a donor/acceptor FRET pair at or near their N/C‐termini. Time‐resolved FRET (tr‐FRET) analysis revealed that the peptide corresponding the C‐terminal region of SOD1 forms urea sensitive structure. Utilizing 2‐dimensional Maximum Entropy Modeling (MEM), we found that this peptide undergoes a discrete transition between a compact state and a random coil‐like structure. Mutations show this collapse results from a non‐native intramolecular interaction involving a hydrophobic sequence at the C‐terminus as well as a polar/charged sequence at the N‐terminus. Ongoing studies are examining the behavior of these segments in the full‐length protein with tr‐FRET. These results are consistent with the results of proteolysis and crystallographic studies from other labs and together suggest that this region of SOD1 may present a potential target for novel therapeutics to treat ALS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Slow Interconversion in a Conformationally Heterogeneous Unfolded-State Ensemble of Outer Membrane Phospholipase A

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of Outer Membrane Phospholipase A (OmpLA) to provide a detailed view of its structural dynamics from nanoseconds to milliseconds. We find that, even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states as compared with a random-coil polypeptide may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.

In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides.

Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models.

Slow Interconversion in a Heterogeneous Unfolded-State Ensemble of Outer-Membrane Phospholipase A

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of outer-membrane phospholipase A (OmpLA) to provide a detailed view of its structural dynamics on timescales from nanoseconds to milliseconds. We find that even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain-reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states, as compared with a random-coil polypeptide, may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.

Effect of urea on heat-induced gelation of bovine serum albumin (BSA) studied by rheology and small angle neutron scattering (SANS)

This paper reports the effects of urea on the heat-induced gelation of bovine serum albumin (BSA), which was studied by the tube inversion method, rheological measurements, and small-angle neutron scattering (SANS). An increase in the urea concentration accelerated the rate of gelation because the protein molecules have already been unfolded to some extent during sample preparation in the urea solution. In addition, the BSA solution in the presence of urea underwent a sol-gel-sol transition during the time sweep test at a constant temperature of 80oC. On the other hand, the BSA solution without urea turned into a hard and brittle gel that did not return to the solution state during isothermal heating at a constant temperature of 80oC. Aggregation and re-bonding of the denatured and unfolded protein chains led to gel formation. Urea added to the protein denatures its tertiary and secondary structures by simultaneously disrupting the hydrogen bonds, hydrophobic interactions, and altering the solvent properties. Furthermore, urea induces thermoreversible chemical interactions in BSA solutions leading to the formation of a gel with dynamic properties under these experimental conditions.

ERAP1 deficiency mitigates HLA-B27 misfolding and ER stress, and protects HLA-B27 transgenic rats from arthritis and orchitis

Abstract Endoplasmic reticulum aminopeptidase 1 (ERAP1) is associated with ankylosing spondylitis (AS) in HLA-B27-positive individuals. ERAP1 optimizes peptide length for binding to MHC class I molecules. However, the role of HLA-B27-bound peptides in AS pathogenesis remains unclear. We investigated epistasis between ERAP1 and HLA-B27 in a rat model of spondyloarthritis (SpA). ERAP1-deficient rats generated by TALEN-mediated genome editing were crossed with disease prone HLA-B27 transgenic (B27 Tg) rats (33-3 transgene). ERAP1 deficiency reduced the prevalence of arthritis (4/34 in ERAP1−/− vs. 12/37 in ERAP1+/+; p&amp;lt;0.05) and testicular inflammation (6/34 vs. 15/37, p&amp;lt;0.05) in male B27 Tg rats. Wild type and HLA-B7 transgenic (B7 Tg) rats with and without ERAP1 remained healthy. Bone marrow-derived macrophages (BMDMs) from B27 Tg ERAP1−/− rats exhibited a 12-fold (p&amp;lt;0.05) increase in MARB4-reactive cell surface B27 complexes containing long peptides, a 30% reduction of total cellular unfolded B27 heavy chains (HC) (p&amp;lt;0.01), and a 32% increase in the ratio of folded/unfolded B27 HC (p=0.01). ERAP1 deficiency also reduced by 50% the formation of disulfide-linked dimers and B27-BiP complexes (p&amp;lt;0.01). ERAP1-deficiency attenuated BiP and CHOP induction and XBP1 splicing (50%, 27%, and 25%, respectively; p&amp;lt;0.01) caused by IFNg-induced B27 upregulation. These results indicate that ERAP1-deficiency is protective for arthritis development in 33-3 B27 Tg rats, and raise the possibility that alleviation of ER stress may contribute to protection, although further investigation is needed to define the mechanism.

The push-and-pull hypothesis in protein unfolding, misfolding and aggregation

The combination of biophysical and structural techniques has allowed the visualization of species classified as dry molten-globule states. Further destabilization causes these structures to follow through a wet-globule stage to reach an unfolded chain. We have recently combined small angle X-ray scattering and nuclear magnetic resonance to observe these species, and we introduce a push-and-pull hypothesis to explain the dissimilar actions of urea and high pressure on proteins. The implications of these molten-globule states are further discussed in light of their potential physiological and pathological roles, especially in protein misfolding diseases.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold’s regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cellremoving detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology.Statement of SignificancePreservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.