scholarly journals Structural dissection of alkaline-denatured pepsin

2004 ◽  
Vol 18 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Yuji O. Kamatari ◽  
Christopher M. Dobson ◽  
Takashi Konno
Keyword(s):  
Limited Proteolysis ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Core Region ◽  
Detailed Knowledge ◽  
Native State ◽  
Denatured State ◽  
Residual Structure ◽  
Alkaline Denaturation ◽  
Folded Core

Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IPstate) is composed of a compact C‒terminal lobe and a largely unstructured N‒terminal lobe. In the present study, we have investigated the residual structure in the IPstate in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‒terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‒terminal lobe contributes a substantial amount of additional residual structure to the IPstate of pepsin. CD spectra indicate in addition that significant non‒native α-helical structure is present in the C‒terminal lobe of the structure when the N‒terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the IPstate is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.

scholarly journals The Conformational Stabilities of Tropomyosins

1976 ◽  
Vol 29 (6) ◽  
pp. 405 ◽  
Author(s):  
EF Woods
Keyword(s):  
Guanidine Hydrochloride ◽  
Coiled Coil ◽  
Helical Structure ◽  
Globular Proteins ◽  
Free Energies ◽  
Native State ◽  
Denatured State ◽  
Partially Unfolded ◽  
The Stability ◽  
Partially Unfolded States

The stability to denaturation by heat and guanidine hydrochloride of seven vertebrate (including skeletal, cardiac and smooth muscle) tropomyosins and three invertebrate tropomyosins was examined. The transition profiles were discontinuous and in many cases distinct plateaux were observed which indicated the presence of unique partially unfolded states at intermediate temperatures and guanidine hydrochloride concentrations. The denaturation by guanidine hydrochloride could be described in the majority of cases by a model in which the native state unfolds to a partially unfolded stable intermediate which then unfolds to the completely denatured state. On this basis it was possible to estimate the free energies of unfolding in water. It was shown that part of the IX-helical structure of tropomyosin is only marginally stable and the free energy of unfolding in water of this segment is less than values found for globular proteins, whereas another segment (or segments) has a stability comparable to that found for globular proteins. The stepwise unfolding may be explained in terms of the coiled-coil interactions in tropomyosin.

scholarly journals Understanding How Staphylococcal Autolysin Domains Interact With Polystyrene Surfaces

2021 ◽  
Vol 12 ◽  
Author(s):  
Radha P. Somarathne ◽  
Emily R. Chappell ◽  
Y. Randika Perera ◽  
Rahul Yadav ◽  
Joo Youn Park ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Medical Devices ◽  
Biofilm Formation ◽  
Limited Proteolysis ◽  
Cd Spectroscopy ◽  
Bacterial Attachment ◽  
Binding Interaction ◽  
Biofilm Growth ◽  
Source Of Infection ◽  
Structural Details

Biofilms, when formed on medical devices, can cause malfunctions and reduce the efficiency of these devices, thus complicating treatments and serving as a source of infection. The autolysin protein of Staphylococcus epidermidis contributes to its biofilm forming ability, especially on polystyrene surfaces. R2ab and amidase are autolysin protein domains thought to have high affinity to polystyrene surfaces, and they are involved in initial bacterial attachment in S. epidermidis biofilm formation. However, the structural details of R2ab and amidase binding to surfaces are poorly understood. In this study, we have investigated how R2ab and amidase influence biofilm formation on polystyrene surfaces. We have also studied how these proteins interact with polystyrene nanoparticles (PSNPs) using biophysical techniques. Pretreating polystyrene plates with R2ab and amidase domains inhibits biofilm growth relative to a control protein, indicating that these domains bind tightly to polystyrene surfaces and can block bacterial attachment. Correspondingly, we find that both domains interact strongly with anionic, carboxylate-functionalized as well as neutral, non-functionalized PSNPs, suggesting a similar binding interaction for nanoparticles and macroscopic surfaces. Both anionic and neutral PSNPs induce changes to the secondary structure of both R2ab and amidase as monitored by circular dichroism (CD) spectroscopy. These changes are very similar, though not identical, for both types of PSNPs, suggesting that carboxylate functionalization is only a small perturbation for R2ab and amidase binding. This structural change is also seen in limited proteolysis experiments, which exhibit substantial differences for both proteins when in the presence of carboxylate PSNPs. Overall, our results demonstrate that the R2ab and amidase domains strongly favor adsorption to polystyrene surfaces, and that surface adsorption destabilizes the secondary structure of these domains. Bacterial attachment to polystyrene surfaces during the initial phases of biofilm formation, therefore, may be mediated by aromatic residues, since these residues are known to drive adsorption to PSNPs. Together, these experiments can be used to develop new strategies for biofilm eradication, ensuring the proper long-lived functioning of medical devices.

scholarly journals Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners

2021 ◽  
Vol 22 (19) ◽  
pp. 10805
Author(s):  
José L. Neira ◽  
David Ortega-Alarcón ◽  
Bruno Rizzuti ◽  
Martina Palomino-Schätzlein ◽  
Adrián Velázquez-Campoy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Antibacterial Properties ◽  
Helical Structure ◽  
Titration Calorimetry ◽  
Its Sequence ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Residual Structure ◽  
Α Helix

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

scholarly journals Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

2021 ◽  
Vol 119 (1) ◽  
pp. e2109169119
Author(s):  
Kristen A. Gaffney ◽  
Ruiqiong Guo ◽  
Michael D. Bridges ◽  
Shaima Muhammednazaar ◽  
Daoyang Chen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Limited Proteolysis ◽  
Water Soluble ◽  
Disordered Proteins ◽  
Resonance Spectroscopy ◽  
Denatured State ◽  
E Coli ◽  
State Ensemble

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol

2006 ◽  
Vol 84 (2) ◽  
pp. 126-134 ◽  
Author(s):  
Fouzia Rashid ◽  
Sandeep Sharma ◽  
M A Baig ◽  
Bilqees Bano
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Molten Globule ◽  
Structural Features ◽  
Cd Spectroscopy ◽  
Native State ◽  
Molten Globule State ◽  
Fluorescence Measurements ◽  
Helical Content ◽  
Human Placental Cystatin

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of α-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.Key words: human placental cystatin, molten globule, acid-induced state, trifluoroethanol, methanol, CD spectroscopy, ANS fluorescence, pH, protein folding.

The Integrity of the Acromioclavicular Capsule Ensures Physiological Centering of the Acromioclavicular Joint Under Rotational Loading

2018 ◽  
Vol 46 (6) ◽  
pp. 1432-1440 ◽  
Author(s):  
Felix G.E. Dyrna ◽  
Florian B. Imhoff ◽  
Andreas Voss ◽  
Sepp Braun ◽  
Elifho Obopilwe ◽  
...  
Keyword(s):  
Translational Motion ◽  
Measuring System ◽  
Resistance Force ◽  
Materials Testing ◽  
Detailed Knowledge ◽  
Native State ◽  
Ac Joint ◽  
Axial Forces ◽  
Resistance Torque ◽  
Posterior Translation

Background: The acromioclavicular (AC) capsule is an important stabilizer against horizontal translation and also contributes to the strut function of the clavicle, which guides rotation of the scapula. To best reproduce the biomechanical properties and the complex 3-dimensional (3D) guidance of the AC joint, detailed knowledge of the contribution of each of the distinctive capsular structures is needed. Purpose/Hypothesis: To perform a detailed biomechanical evaluation of the specific capsular structures of the AC joint and their contribution to translational and rotational stability. The hypothesis was that successive cutting of each quadrant of the AC capsule would result in increased instability and increased amplitude of the clavicle’s motion in relation to the acromion. Study Design: Controlled laboratory study. Methods: Thirty-two fresh-frozen human cadaveric shoulders were used. Each scapula was fixed to a swivel fixture of a servohydraulic materials testing system. The AC capsule was dissected in serial steps with immediate rotational and horizontal testing after each cut. A 3D optical measuring system was used to evaluate 3D movement. Posterior translation, rotation, and displacement of the lateral clavicle in relation to the center of rotation were measured. Torques and axial forces required to rotate and translate the clavicle were recorded. Results: When posterior translational force was applied, all specimens with a completely cut AC capsule demonstrated a significant loss of resistance force against the translational motion when compared with the native state ( P < .05). The resistance force against posterior translation was reduced to less than 27% of the native state for all specimens. Sequential cutting of the AC capsule resulted in a significant reduction of resistance torque against anterior rotation for all specimens with less than 22% of resistance force compared with the native state. Cutting 50% of the capsule reduced the resistance torque for all segments and all testing modalities (posterior translation as well as anterior and posterior rotation) significantly compared with the native state ( P < .05). Cutting the entire AC capsule resulted in a significant increase in motion within the joint as a sign of decentering of the AC joint when torque was applied. All groups demonstrated a significant increase of motion in all directions when the AC capsule was cut by 50%. Conclusion: Cutting the entire capsule (with intact coracoclavicular [CC] ligaments) reduced the resistance force to less than 25% compared with the native state during translational testing and less than 10% compared with the native state during rotational testing. However, the anterior segments of the capsule provided the greatest stability under rotational loading. Second, the amplitude of the joint’s motion significantly increased under rotational stress, indicating increased amplitude of the clavicle’s motion in relation to the acromion when the ligamentous structures of the AC capsule are dissected. Clinical Relevance: To best restore stability to the AC joint, the relevance and function of each section of the circumferential AC capsule need to be understood. Our findings support the synergistic contribution of the CC ligaments and AC capsular structures to AC joint stability. This synergy supports the need to address both structures to achieve anatomic reconstruction.

Folding mechanism of the C‐terminal domain of nucleophosmin: residual structure in the denatured state and its pathophysiological significance

2009 ◽  
Vol 23 (8) ◽  
pp. 2360-2365 ◽  
Author(s):  
Flavio Scaloni ◽  
Stefano Gianni ◽  
Luca Federici ◽  
Brunangelo Falini ◽  
Maurizio Brunori
Keyword(s):  
Denatured State ◽  
Residual Structure ◽  
Folding Mechanism ◽  
Terminal Domain

Collagen structure: new tricks from a very old dog

2016 ◽  
Vol 473 (8) ◽  
pp. 1001-1025 ◽  
Author(s):  
Jordi Bella
Keyword(s):  
Molecular Conformation ◽  
Helical Structure ◽  
Molecular Engineering ◽  
Detailed Knowledge ◽  
Collagen Structure ◽  
Nmr Studies ◽  
Conformational Variability ◽  
Collagen Peptides ◽  
Triple Helical Domain ◽  
Model Peptides

The main features of the triple helical structure of collagen were deduced in the mid-1950s from fibre X-ray diffraction of tendons. Yet, the resulting models only could offer an average description of the molecular conformation. A critical advance came about 20 years later with the chemical synthesis of sufficiently long and homogeneous peptides with collagen-like sequences. The availability of these collagen model peptides resulted in a large number of biochemical, crystallographic and NMR studies that have revolutionized our understanding of collagen structure. High-resolution crystal structures from collagen model peptides have provided a wealth of data on collagen conformational variability, interaction with water, collagen stability or the effects of interruptions. Furthermore, a large increase in the number of structures of collagen model peptides in complex with domains from receptors or collagen-binding proteins has shed light on the mechanisms of collagen recognition. In recent years, collagen biochemistry has escaped the boundaries of natural collagen sequences. Detailed knowledge of collagen structure has opened the field for protein engineers who have used chemical biology approaches to produce hyperstable collagens with unnatural residues, rationally designed collagen heterotrimers, self-assembling collagen peptides, etc. This review summarizes our current understanding of the structure of the collagen triple helical domain (COL×3) and gives an overview of some of the new developments in collagen molecular engineering aiming to produce novel collagen-based materials with superior properties.

scholarly journals A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

2016 ◽  
Vol 84 (9) ◽  
pp. 2662-2670 ◽  
Author(s):  
Christian González-Rivera ◽  
Anne M. Campbell ◽  
Stacey A. Rutherford ◽  
Tasia M. Pyburn ◽  
Nora J. Foegeding ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Limited Proteolysis ◽  
Helical Structure ◽  
Water Soluble ◽  
Host Cells ◽  
Mutant Form ◽  
Terminal Portion ◽  
Toxic Activity ◽  
Content Type ◽  
Amino Terminal

Helicobacter pylorisecretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted byH. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.

The Denatured States of Lysozyme

1973 ◽  
Vol 51 (5) ◽  
pp. 581-585 ◽  
Author(s):  
M. Kugimiya ◽  
C. C. Bigelow
Keyword(s):  
Lithium Perchlorate ◽  
Difference Spectroscopy ◽  
Hen Egg White Lysozyme ◽  
Guanidinium Chloride ◽  
Denatured State ◽  
Residual Structure ◽  
Egg White Lysozyme ◽  
Temperature State ◽  
Hen Egg White ◽  
Denatured States

The denaturation of hen egg-white lysozyme has been studied under a variety of denaturing conditions by difference spectroscopy, polarimetry, and viscometry. It has been found that the lysozyme molecule can take up four different denatured conformations, depending on the denaturant used. Urea and guanidinium chloride produce the most completely denatured state, IV, and, listed in the order of increasing residual structure, lithium chloride gives state III, temperature state II, and lithium perchlorate state I. Differences in the spectra at 300 nm have been a valuable aid in relating the different denatured states to each other.

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