A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4.

Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix-helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.

Self-Assembly of Exendin-4-Derived Dual Peptide Agonists is Mediated by Acylation and Correlated to the Length of Conjugated Fatty Acyl Chains.

Dual glucagon-like peptide-1/glucagon receptor agonists have emerged as promising candidates for the treatment of diabetes and obesity. Issues of degradation sensitivity and rapid renal clearance are addressed, for example, by the conjugation of peptides to fatty acid chains, promoting reversible albumin binding. We use combined dynamic and static light scattering to directly measure the self-assembly of a set of dual peptide agonists based on the exendin-4 structure with varying fatty acid chain lengths in terms of apparent molecular mass and hydrodynamic radius (RS). We use NMR spectroscopy to gain an insight into the molecular architecture of the assembly. We investigate conformational changes of the monomeric subunits resulting from peptide self-assembly and assembly stability as a function of the fatty acid chain length using circular dichroism and fluorescence spectroscopy. Our results demonstrate that self-assembly of the exendin-4-derived dual agonist peptides is essentially driven by hydrophobic interactions involving the conjugated acyl chains. The fatty acid chain length affects assembly equilibria and the assembly stability, although the peptide subunits in the assembly retain a dynamic secondary structure. The assembly architecture is characterized by juxtaposition of the fatty acyl side chains and a hydrophobic cluster of the peptide moiety. This cluster experiences local conformational changes in the assembly compared to the monomeric unit leading to a reduction in solvent exposure. The N-terminal half of the peptide and a C-terminal loop are not in contact with neighboring peptide subunits in the assemblies. Altogether, our study contributes to a thorough understanding of the association characteristics and the tendency toward self-assembly in response to lipidation. This is important not only to achieve the desired bioavailability but also with respect to the physical stability of peptide solutions.

Comment on "Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water".

Riback et al (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experiments to infer a degree of compaction for unfolded proteins in water versus chemical denaturant that is highly consistent with the results from Förster resonance energy transfer (FRET) experiments. There is thus no "contradiction" between the two methods, nor evidence to support their claim that commonly used FRET fluorophores cause protein compaction.

Rapid-Acting and Human Insulins: Hexamer Dissociation Kinetics upon Dilution of the Pharmaceutical Formulation.

PURPOSE: Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. METHODS: Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. RESULTS: Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. CONCLUSION: Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods.

There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in Escherichia coli.

The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.

IR spectroscopic analyses of amyloid fibril formation of ß2-microglobulin using a simplified procedure for its in vitro generation at neutral pH.

ß2-microglobulin (ß2m) is known to be the major component of fibrillar deposits in the joints of patients suffering from dialysis-related amyloidosis. We have developed a simplified procedure to convert monomeric recombinant ß2m into amyloid fibrils at physiological pH by a combination of stirring and heating, enabling us to follow conformational changes associated with the assembly by infrared spectroscopy and electron microscopy. Our studies reveal that fibrillogenesis begins with the formation of relatively large aggregates, with secondary structure not significantly altered by the stirring-induced association. In contrast, the conversion of the amorphous aggregates into amyloid fibrils is associated with a profound re-organization at the level of the secondary and tertiary structures, leading to non-native like parallel arrangements of the ß-strands in the fully formed amyloid structure of ß2m. This study highlights the power of an approach to investigate the formation of ß2m fibrils by a combination of biophysical techniques including IR spectroscopy.

Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy.

The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution.

Dynamic and static light scattering of intrinsically disordered proteins.

Molecular parameters such as size, molar mass, and intermolecular interactions, which are important to identify and characterize intrinsically disordered proteins (IDPs), can be obtained from light scattering measurements. In this chapter, we discuss the physical basis of light scattering, experimental techniques, sample treatment, and data evaluation with special emphasis on studies on proteins. Static light scattering (SLS) is capable of measuring molar masses within the range 10(3)-10(8) g/mol and is therefore ideal for determining the state of association of proteins in solution. Since proteins are in general too small to obtain the geometric radius of gyration R (G) from SLS, it is more useful to determine the hydrodynamic Stokes radius, R (S), which can be obtained easily and quickly from dynamic light scattering (DLS) experiments. Accordingly, DLS is an appropriate technique to monitor expansion or compaction of protein molecules. This is especially important for IDPs, which can be recognized and characterized by comparing the measured Stokes radii with those calculated for particular reference states, such as the compactly folded and the fully unfolded states. The combined application of DLS and SLS improves measurements of the molar mass and is essential when changes in the molecular dimensions and molecular association/dissociation take place simultaneously.

Three-dimensional mapping of the B1 field using an optimized phase-based method: application to hyperpolarized 3He in lungs.

A novel method is presented for the three-dimensional mapping of the B(1) -field of a transmit radio-frequency MR coil. The method is based on the acquisition of phase images, where the effective flip angle is encoded in the phase of the nonselective hard pulse excitation. The method involves the application of a rectangular composite pulse as excitation in a three-dimensional gradient recall echo to produce measurable phase angle variation. However, such a pulse may significantly increase the radio-frequency power deposition in excess of the standard acceptable SAR limits, imposing extremely long TRs (>100 msec), which would result in acquisition times significantly greater than a single breath-hold. In this study, the phases of the radio-frequency excitation are modified, resulting in a different pulse sequence scheme. It is shown that the new method increases sensitivity with respect to radio-frequency inhomogeneities by up to 10 times, and reduces the total duration of the pulse so that three-dimensional B(1) mapping is possible with (3) He in lungs within a single breath-hold. Computer simulations demonstrate the increase in sensitivity. Phantom results with (1) H MRI are used for validation. In vivo results are presented with hyperpolarized (3) He in human lungs at 1.5T.

Symptomatic atherosclerotic occlusive disease of all supra-aortic arch vessels treated with total aortic arch rerouting.

We present an uncommon case of symptomatic atherosclerotic occlusive disease of all supra-aortic arch vessels and its surgical treatment by total aortic arch rerouting after endarteriectomy of all target vessels.

Yeast hexokinase isoenzyme ScHxk2: stability of a two-domain protein with discontinuous domains.

The hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2) represents an archetype of a two-domain protein with the active site located in a cleft between the two domains. Binding of the substrate glucose results in a rigid body movement of the two domains leading to a cleft closure of the active site. Both domains of this enzyme are composed of discontinuous peptide sequences. This structural feature is reflected in the stability and folding of the ScHxk2 protein. Structural transitions induced by urea treatment resulted in the population of a thermodynamically stable folding intermediate, which, however, does not correspond to a molecule with one domain folded and the other unfolded. As demonstrated by different spectroscopic techniques, both domains are structurally affected by the partial denaturation. The intermediate possesses only 40% of the native secondary structural content and a substantial increase in the Stokes radius as judged by circular dichroism and dynamic light scattering analyses. One-dimensional ¹H NMR data prove that all tryptophan residues are in a non-native environment in the intermediate, indicating substantial changes in the tertiary structure. Still, the intermediate possesses quite a high stability for a transition intermediate of about ΔG = -22 kJ mol⁻¹.

Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins.

We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin alpha suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.

The oligomerization of the coiled coil-domain of occludin is redox sensitive.

The transmembrane tight junction protein occludin is sensitive to oxidative stress. Occludin oligomerizes; however, its function in the tight junction is unknown. The cytosolic C-terminal tail contains a coiled coil-domain and forms dimers contributing to the oligomerization. The regulation of the oligomerization remains unclear. As the domain area contains sulfhydryl residues, we tested the hypothesis that the dimerization of the coiled coil-domain depends on these residues. We showed that the dimerization is modulated by the thiol concentration in the low-millimolar range, which is relevant both for physiological and pathophysiological conditions. Masking the sulfhydryl residues in the fragment by covalent binding of 4-vinyl pyridine prevented the dimerization but did not affect its helical structure and cylindric shape. The data demonstrate, for the first time, that disulfide bridge formation of murine cystein 408 is involved in the dimerization. This process is redox-sensitive but the secondary structure of the domain is not. It is concluded that the dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions.

Early stages of misfolding and association of beta2-microglobulin: insights from infrared spectroscopy and dynamic light scattering.

Conformational changes associated with the assembly of recombinant beta 2-microglobulin in vitro under acidic conditions were investigated using infrared spectroscopy and static and dynamic light scattering. In parallel, the morphology of the different aggregated species obtained under defined conditions was characterized by electron microscopy. The initial salt-induced aggregate form of beta 2-microglobulin, composed of small oligomers (dimers to tetramers), revealed the presence of beta-strands organized in an intramolecular-like fashion. Further particle growth was accompanied by the formation of intermolecular beta-sheet structure and led to short curved forms. An increase in temperature by only 25 degrees C was able to disaggregate these assemblies, followed by the formation of longer filamentous structures. In contrast, a rise in temperature up to 100 degrees C was associated with a reorganization of the short curved forms at the level of secondary structure and the state of assembly, leading to a species with a characteristic infrared spectrum different from those of all the other aggregates observed before, suggesting a unique overall structure. The infrared spectral features of this species were nearly identical to those of beta 2-microglobulin assemblies formed at low ionic strength with agitation, indicating the presence of fibrils, which was confirmed by electron microscopy. The observed spectroscopic changes suggest that the heat-triggered conversion of the short curved assemblies into fibrils involves a reorganization of the beta-strands from an antiparallel arrangement to a parallel arrangement, with the latter being characteristic of amyloid fibrils of beta 2-microglobulin.

Intrapulmonary 3He gas distribution depending on bolus size and temporal bolus placement.

OBJECTIVE: Dynamic ventilation (3)He-MRI is a new method to assess pulmonary gas inflow. As differing airway diameters throughout the ventilatory cycle can influence gas inflow this study intends to investigate the influence of volume and timing of a He gas bolus with respect to the beginning of the tidal volume on inspiratory gas distribution. MATERIALS AND METHODS: An ultrafast 2-dimensional spoiled gradient echo sequence (temporal resolution 100 milliseconds) was used for dynamic ventilation (3)He-MRI of 11 anesthetized and mechanically ventilated pigs. The applied (3)He gas bolus was varied in volume between 100 and 200 mL. A 150-mL bolus was varied in its application time after the beginning of the tidal volume between 0 and 1200 milliseconds. Signal kinetics were evaluated using an in-house developed software after definition of parameters for the quantitative description of (3)He gas inflow. RESULTS: The signal rise time (time interval between signal in the parenchyma reaches 10% and 90% of its maximum) was prolonged with increasing bolus volume. The parameter was shortened with increasing delay of (3)He application after the beginning of the tidal volume. Timing variation as well as volume variation showed no clear interrelation to the signal delay time 10 (time interval between signal in the trachea reaches 50% of its maximum and signal in the parenchyma reaches 10% of its maximum). CONCLUSIONS: Dynamic ventilation (3)He-MRI is able to detect differences in bolus geometry performed by volume variation. Pulmonary gas inflow as investigated by dynamic ventilation (3)He-MRI tends to be accelerated by an increasing application delay of a (3)He gas bolus after the beginning of the tidal volume.

Oxygen-sensitive 3He-MRI in bronchiolitis obliterans after lung transplantation.

Oxygen-sensitive 3He-MRI was studied for the detection of differences in intrapulmonary oxygen partial pressure (pO2) between patients with normal lung transplants and those with bronchiolitis obliterans syndrome (BOS). Using software developed in-house, oxygen-sensitive 3He-MRI datasets from patients with normal lung grafts (n = 8) and with BOS (n = 6) were evaluated quantitatively. Datasets were acqiured on a 1.5-T system using a spoiled gradient echo pulse sequence. Underlying diseases were pulmonary emphysema (n = 10 datasets) and fibrosis (n = 4). BOS status was verified by pulmonary function tests. Additionally, 3He-MRI was assessed blindedly for ventilation defects. Median intrapulmonary pO2 in patients with normal lung grafts was 146 mbar compared with 108 mbar in patients with BOS. Homogeneity of pO2 distribution was greater in normal grafts (standard deviation pO2 34 versus 43 mbar). Median oxygen decrease rate during breath hold was higher in unaffected patients (-1.75 mbar/s versus -0.38 mbar/s). Normal grafts showed fewer ventilation defects (5% versus 28%, medians). Oxygen-sensitive 3He-MRI appears capable of demonstrating differences of intrapulmonary pO2 between normal lung grafts and grafts affected by BOS. Oxygen-sensitive 3He-MRI may add helpful regional information to other diagnostic techniques for the assessment and follow-up of lung transplant recipients.

Involvement of jugular valve insufficiency in cerebral venous air embolism.

BACKGROUND: Cerebral venous air entrapment is a rare finding on cranial computed tomography (CT) scan. Peripheral air embolism is discussed as a potential cause. However, the mechanism of retrograde passage through internal jugular valves and veins is unclear. CASE REPORT: The case of a patient is reported, who had air entrapment in the left cavernous sinus. Prior to CT scanning, a peripheral intravenous line had been placed. Ultrasound revealed excessive insufficiency of the left internal jugular valve. To further study the mechanism of embolism, an echo contrast agent was injected into the cubital vein. A Valsalva maneuver resulted in retrograde transition of microbubbles across the insufficient valve. Valvular function on the unaffected right side was intact. CONCLUSIONS: This case report gives insight into the mechanism of cerebral venous air embolism. This is the firstcase describing jugular valve insufficiency as the missing link between peripheral air embolism and cerebral venous air entrapment.

Clinical aspects of the apparent diffusion coefficient in 3He MRI: results in healthy volunteers and patients after lung transplantation.

PURPOSE: To measure the apparent diffusion coefficient (ADC) after inhalation of hyperpolarized (3)He in healthy volunteers and lung transplant recipients, and demonstrate the gravity dependence of ADC values. MATERIALS AND METHODS: Six healthy volunteers, 10 patients after single-lung transplantation, and six patients after double-lung transplantation were examined at 1.5T during inspiration and expiration. The inhalation of 300 mL of hyperpolarized (3)He was performed with a computer-controlled delivery device. A two-dimensional fast low-angle shot (FLASH) sequence measured the (3)He diffusive gas movement. From these data the ADC was calculated. RESULTS: The mean ADC was 0.143 cm(2)/second in healthy individuals, 0.162 cm(2)/second in transplanted healthy lungs, and 0.173 cm(2)/second in rejected transplanted lungs, whereas it was 0.216 cm(2)/second in native fibrotic lungs and 0.239 cm(2)/second in emphysematous lungs. The difference in mean ADC values among healthy lungs, healthy transplanted lungs, and native diseased lungs was significant (P < 0.001). In inspiration the healthy volunteers showed higher ADC values in the anterior than in the posterior parts of the lungs. In expiration this gradient doubled. CONCLUSION: An anterior-posterior (A/P) gradient was found in inspiration and expiration in healthy lungs. Healthy, transplanted, and native diseased lungs had significantly different mean ADC values. From our preliminary results, (3)He MRI appears to be sensitive for detecting areas of abnormal ventilation in transplanted lungs.

Chronic thromboembolic pulmonary hypertension - assessment by magnetic resonance imaging.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a severe disease that has been ignored for a long time. However, with the development of improved therapeutic modalities, cardiologists and thoracic surgeons have shown increasing interest in the diagnostic work-up of this entity. The diagnosis and management of chronic thromboembolic pulmonary hypertension require a multidisciplinary approach involving the specialties of pulmonary medicine, cardiology, radiology, anesthesiology and thoracic surgery. With this approach, pulmonary endarterectomy (PEA) can be performed with an acceptable mortality rate. This review article describes the developments in magnetic resonance (MR) imaging techniques for the diagnosis of chronic thromboembolic pulmonary hypertension. Techniques include contrast-enhanced MR angiography (ce-MRA), MR perfusion imaging, phase-contrast imaging of the great vessels, cine imaging of the heart and combined perfusion-ventilation MR imaging with hyperpolarized noble gases. It is anticipated that MR imaging will play a central role in the initial diagnosis and follow-up of patients with CTEPH.