Folding Phase Research Articles

Tectonic evolution of the Yaoundé segment of the Neoproterozoic Central African Orogenic Belt in southern Cameroon

The deformation history of the Neoproterozoic Central African Orogenic Belt in southern Cameroon is well recorded in the low- to high-grade rocks outcropping in the area around Yaoundé. The fabrics in these rocks are consistent with two main ductile deformation events D1 and D2. D1 predated emplacement of calc-alkaline dioritic bodies and caused the formation of nappes that resulted in high-pressure granulite metamorphism of soft sediments. A strong overprinting of these nappes during D2 symmetric extension, probably associated with large-scale foliation boudinage and (or) gneissic doming and intense magmatic underplating, gave rise to regional flat-lying fabrics. The latter were further buckled by D3 and D4 folding phases defining a vertical constriction occurring with a major east–west to NW–SE shortening direction. The corresponding F3 and F4 folds trend north–south to NE–SW and east–west to NW–SE, respectively, and represent the main regional strain patterns. Based on the east–west to NW–SE maximum shortening orientation indicated by F3 folds, it is proposed that the nappe-stacking phase D1 occurred in the same direction. The deformation history in the area can thus be described as corresponding principally to alternating east–west to NW–SE contractions and north–south to NE–SW orogenic-parallel extensions. At the regional scale, this could be due to the Transaharan east–west collisional system.

A scFv Antibody Mutant Isolated in a Genetic Screen for Improved Export via the Twin Arginine Transporter Pathway Exhibits Faster Folding

Proteins destined for export across the cytoplasmic membrane via the post-translational Sec-dependent route have to be maintained in a largely unfolded state within the cytoplasm. In sharp contrast, only proteins that have folded into a native-like state within the cytoplasm are competent for export via the twin arginine translocation (Tat) pathway. Proteins that contain disulfide bonds, such as scFv antibody fragments, can be translocated via Tat only when expressed in Escherichia coli trxB gor mutant strains having an oxidizing cytoplasm. However, export is poor with the majority of the protein accumulating in the cytoplasm and only a fraction exported to the periplasmic space. Using a high throughput fluorescence screen, we isolated a mutant of the anti-digoxin 26-10 scFv from a large library of random mutants that is exported with a higher yield into the periplasm. In vitro refolding experiments revealed that the mutant scFv exhibits a 250% increase in the rate constant of the critical second phase of folding. This result suggests that Tat export competence is related to the protein folding rate and could be exploited for the isolation of faster folding protein mutants.

Rapid Folding and Unfolding of Apaf-1 CARD

Caspase recruitment domains (CARDs) are members of the death domain superfamily and contain six antiparallel helices in an α-helical Greek key topology. We have examined the equilibrium and kinetic folding of the CARD of Apaf-1 (apoptotic protease activating factor 1), which consists of 97 amino acid residues, at pH 6 and pH 8. The results showed that an apparent two state equilibrium mechanism is not adequate to describe the folding of Apaf-1 CARD at either pH, suggesting the presence of intermediates in equilibrium unfolding. Interestingly, the results showed that the secondary structure is less stable than the tertiary structure, based on the transition mid-points for unfolding. Single mixing and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. Kinetic simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. This is in contrast to other CARDs in which folding appears to be dominated by formation of kinetic traps.

Tectono‐metamorphic history of the Tacagua ophiolitic unit (Cordillera de la Costa, northern Venezuela): Insights in the evolution of the southern margin of the Caribbean Plate

Abstract The southern margin of the Caribbean Plate is well exposed in the Cordillera de la Costa of northern Venezuela, where amalgamated terranes consisting of continental and oceanic units occur. In the Cordillera de la Costa, metamorphosed oceanic units crop out along the coast near Caracas. Among them, the Tacagua unit is characterized by metaserpentinites and metabasites showing mid‐oceanic ridge basalt geochemical affinity. These lithologies, representative of a disrupted ophiolite sequence, are associated with metasediments consisting of calcschists alternating with pelitic and psammitic schists, whose protoliths were probably represented by deep‐sea hemipelagic and turbiditic deposits. In the Tacagua unit, a polyphase deformation history has been reconstructed, consisting of four folding phases from D1 to D4. Geological setting suggests an involvement of the Tacagua unit in the processes connected with a subduction zone. The following deformations (from D2 to D4) observed in the field might be related to the exhumation history of the Tacagua unit. The late deformation history consists of an alternation of deformation phases characterized by displacement parallel (D2 and D4 phases) and normal (D3 phase) to plate boundary between the Caribbean and South America Plates. All lines of geological evidence suggest that the whole evolution of the Tacagua unit was acquired in a setting dominated by oblique convergence, in which alternation of strike‐slip and pure compressional or pure extensional tectonics occurred through time.

Stabilizing IκBα by “Consensus” Design

IκBα is the major regulator of transcription factor NF-κB function. The ankyrin repeat region of IκBα mediates specific interactions with NF-κB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-κB. Using chemical denaturation, we show here that IκBα displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the “consensus” ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IκBα and the stabilized mutants by H/ 2H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

Correlation of folding kinetics with the number and isomerization states of prolines in three homologous proteins of the RNase family

Several studies attribute the slower phases in protein folding to prolyl isomerizations, and several others do not. A correlation exists between the number of prolines in a protein and the complexity of the mechanism with which it folds. In this study, we have demonstrated a direct correlation between the number of cis-prolyl bonds in a native protein and the complexity with which it folds via slower phases by studying the folding of three structurally homologous proteins of the ribonuclease family, namely RNase A, onconase and angiogenin, which differ in the number and isomerization states of their proline residues.

Early Intermediate in Human Prion Protein Folding As Evidenced by Ultrarapid Mixing Experiments

An important step toward understanding the mechanism of the PrP(C)-to-PrP(Sc) conversion is to elucidate the folding pathway(s) of the prion protein. On the basis of stopped-flow measurements, we recently proposed that the prion protein folds via a transient intermediate formed on the submillisecond time scale, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. Here, we have extended these studies to continuous-flow measurements using a capillary mixing system with a time resolution of approximately 100 micros. This allowed us to directly observe two distinct phases in folding of the recombinant human prion protein 90-231, providing unambiguous evidence for rapid accumulation of an early intermediate (with a time constant of approximately 50 micros), followed by a rate-limiting folding step (with a time constant of approximately 700 micros). The present study also clearly demonstrates that the population of the intermediate is significantly increased at mildly acidic pH and in the presence of urea. A similar three-state folding behavior was observed for the Gerstmann-Straussler-Scheinker disease-associated F198S mutant, in which case the population of an intermediate was greatly increased as compared to that of the wild-type protein. Overall, the present data strongly suggest that this partially structured intermediate may be a direct monomeric precursor of the misfolded PrP(Sc) oligomer.

Syn-folding remagnetization of Cambro-Ordovician carbonates from the Pennsylvania Salient post-dates oroclinal rotation

Paleomagnetic directions of 35 sites of Cambro-Ordovician carbonates from 10 anticlines were analyzed to test models of curvature along the Pennsylvania Salient of the Appalachians and to constrain the relative timing of magnetization acquisition. The sites yield directions of magnetization that are almost all reversed with near-horizontal inclinations upon appropriate structural correction. The common, Late Paleozoic (Kiaman-aged) direction and incremental fold tests show that these directions represent remagnetizations carried by authigenic magnetite, acquired just before or during the earlier phases of folding. No convincing indications were found of primary magnetizations. Mean declinations from the northeastern and southwestern limbs of the salient differ by a few degrees, indicating negligible, if any, rotation between the limbs. The results are similar to prior studies of overlying Siluro-Devonian carbonates, showing coherent behavior of the entire Paleozoic cratonic cover. We conclude that the statistically negligible difference in declination indicates that (previously demonstrated) oroclinal bending occurred before carbonates of the Paleozoic stratigraphic cover were remagnetized during the Permian and before regional folding was completed.

Population of On-pathway Intermediates in the Folding of Ubiquitin

The role that intermediate states play in protein folding is the subject of intense investigation and in the case of ubiquitin has been controversial. We present fluorescence-detected kinetic data derived from single and double mixing stopped-flow experiments to show that the F45W mutant of ubiquitin (WT*), a well-studied single-domain protein and most recently regarded as a simple two-state system, folds via on-pathway intermediates. To account for the discrepancy we observe between equilibrium and kinetic stabilities and m-values, we show that the polypeptide chain undergoes rapid collapse to an intermediate whose presence we infer from a fast lag phase in interrupted refolding experiments. Double-jump kinetic experiments identify two direct folding phases that are not associated with slow isomerisation reactions in the unfolded state. These two phases are explained by kinetic partitioning which allows molecules to reach the native state from the collapsed state via two possible competing routes, which we further examine using two destabilised ubiquitin mutants. Interrupted refolding experiments allow us to observe the formation and decay of an intermediate along one of these pathways. A plausible model for the folding pathway of ubiquitin is presented that demonstrates that obligatory intermediates and/or chain collapse are important events in restricting the conformational search for the native state of ubiquitin.

The Acadian fold belt in the Meguma Terrane, Nova Scotia: Cross sections, fold mechanisms, and tectonic implications

Structural data and cross sections from the eastern part of the Devonian‐Carboniferous fold belt of the Meguma Terrane give insight into its structural development and crustal tectonics. The sandy Goldenville Formation, the active competent member during buckling, formed a multilayer several kilometers thick that lay beneath the denser, softer muds and silts of the Halifax Formation. Cross sections reveal 2 orders of folds: 11–18 km wavelength folds are interpreted as resulting from buckling under the influence of gravity; 4–6 km wavelength folds reflect the thickness of the multilayer with buckle shortening in the range 32–44%. Depth to detachment derived from the sections (11–13.5 km below present erosion surface) is such that given the depth of the detachment below the paleosurface during the main phase (pre‐Devonian granite) of folding, it would likely have been a relatively soft and thick shear zone that would have influenced profile geometry of the main phase folds at this stage of fold belt development. Granite production that may have been triggered by mantle delamination during continuing convergence accompanying terminal Pangean assembly and ocean basin closure was followed by rapid exhumation. Later localized deformation (Late Devonian–Carboniferous) in the fold belt took place as the Meguma Terrane was displaced along the terrane‐bounding Cobequid‐Chedabucto fault system. The characteristic asymmetric cross sections of structures of this phase are attributed to cooling and strengthening of the detachment that was a consequence of the postgranite uplift and exhumation.

A study on an axial crush configuration response of thin-wall, steel box components: The quasi-static experiments

An experimental investigation was performed to study a specific axial crush configuration response of steel, square box components under quasi-static testing conditions. For a specific cross-sectional geometry/fabrication process, test specimens were obtained from commercially produced, welded tube lengths of ASTM A36 and ASTM A513 Type 1 plain low-carbon steels and AISI 316 and AISI 304 austenitic stainless steels. Removable grooved caps were used to constrain tube test specimen ends, and collapse initiators in the form of shallow machined grooves were used to control the initial transverse deformations of the test specimen sidewalls. The progressive plastic deformation for all of the test specimens was restricted to the prototype configuration response (fold formation process and the corresponding axial load-axial displacement curve shape) of the symmetric axial crush mode. Crush characteristics were evaluated and, for each material type, observed differences were less than 7% for maximum and minimum load magnitudes and less than 2% for energy absorption, displacement, and mean load quantities in both the initial phase and the secondary folding phase cycles. Overall, results of the study indicate that for a significant range of material strengths, a controlled and repeatable energy absorption process can be obtained for commercially produced steel box components undergoing symmetric axial crush response.

Consequences of ERp57 Deletion on Oxidative Folding of Obligate and Facultative Clients of the Calnexin Cycle

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.

Woes of proline: A cautionary kinetic tale

Woes of proline: A cautionary kinetic tale

Mineralogical and stable isotope studies of gold–arsenic mineralisation in the Sams Creek peralkaline porphyritic granite, South Island, New Zealand

At Sams Creek, a gold-bearing, peralkaline granite porphyry dyke, which has a 7 km strike length and is up to 60 m in thickness, intrudes camptonite lamprophyre dykes and lower greenschist facies metapelites and quartzites of the Late Ordovician Wangapeka formation. The lamprophyre dykes occur as thin (< 3 m) slivers along the contacts of the granite dyke. δ18Omagma values (+5 to +8‰, VSMOW) of the A-type granite suggest derivation from a primitive source, with an insignificant mature crustal contribution. Hydrothermal gold–sulphide mineralisation is confined to the granite and adjacent lamprophyre; metapelite country rocks have only weak hydrothermal alteration. Three stages of hydrothermal alteration have been identified in the granite: Stage I alteration (high fO2) consisting of magnetite–siderite±biotite; Stage II consisting of thin quartz–pyrite veinlets; and Stage III (low fO2) consisting of sulphides, quartz and siderite veins, and pervasive silicification. The lamprophyre is altered to an ankerite–chlorite–sericite assemblage. Stage III sulphide veins are composed of arsenopyrite + pyrite ± galena ± sphalerite ± gold ± chalcopyrite ± pyrrhotite ± rutile ± graphite. Three phases of deformation have affected the area, and the mineralised veins and the granite and lamprophyre dykes have been deformed by two phases of folding, the youngest of which is Early Cretaceous. Locally preserved early-formed fluid inclusions are either carbonic, showing two- or three-phases at room temperature (liquid CO2-CH4 + liquid H2O ± CO2 vapour) or two-phase liquid-rich aqueous inclusions, some of which contain clathrates. Salinities of the aqueous inclusions are in the range of 1.4 to 7.6 wt% NaCl equiv. Final homogenisation temperatures (Th) of the carbonic inclusions indicate minimum trapping temperatures of 320 to 355°C, which are not too different from vein formation temperatures of 340–380°C estimated from quartz–albite stable isotope thermometry. δ18O values of Stage II and III vein quartz range from +12 and +17‰ and have a bimodal distribution (+14.5 and +16‰) with Stage II vein quartz accounting for the lower values. Siderite in Stage III veins have δ18O (+12 to +16‰) and δ13C values (−5‰, relative to VPDB), unlike those from Wangapeka Formation metasediments (δ13Cbulk carbon values of −24 to −19‰) and underlying Arthur Marble marine carbonates (δ18O = +25‰ and δ13C = 0‰). Calculated δ18Owater (+8 to +11‰, at 340°C) and \(\delta^{13}{\text{C}}_{{\rm CO}_{2}}\)(−5‰) values from vein quartz and siderite are consistent with a magmatic hydrothermal source, but a metamorphic hydrothermal origin cannot be excluded. δ34S values of sulphides range from +5 to +10‰ (relative to CDT) and also have a bimodal distribution (modes at +6 and +9‰, correlated with Stage II and Stage III mineralisation, respectively). The δ34S values of pyrite from the Arthur Marble marine carbonates (range from +3 to +13‰) and Wangapeka Formation (range from −4 to +9.5‰) indicate that they are potential sources of sulphur for sulphides in the Sams Creek veins. Another possible source of the sulphur is the lithospheric mantle which has positive values up to +14‰. Ages of the granite, lamprophyre, alteration/mineralisation, and deformation in the region are not well constrained, which makes it difficult to identify sources of mineralisation with respect to timing. Our mineralogical and stable isotope data does not exclude a metamorphic source, but we consider that the source of the mineralisation can best be explained by a magmatic hydrothermal source. Assuming that the hydrothermal fluids were sourced from crystallisation of the Sams Creek granite or an underlying magma chamber, then the Sams Creek gold deposit appears to be a hybrid between those described as reduced granite Au–Bi deposits and alkaline intrusive-hosted Au–Mo–Cu deposits.

Structural evolution and tectonic setting of the Porongos belt, southern Brazil

The SW–NE-striking Porongos belt, located between juvenile Neoproterozoic rocks in the west and the Dom Feliciano belt, characterized by intense reworking of older crust, in the east, comprises a greenschist to amphibolite-facies metavolcano-metasedimentary succession (Porongos sequence) of unknown age with some exposures of Palaeoproterozoic gneisses (Encantadas gneisses). High-temperature ductile deformation of the basement gneisses comprises at least two magmatic events followed by three deformational phases including folding and shearing (DT1–DT3) and can be attributed to the Palaeoproterozoic Trans-Amazonian orogeny. The deformation of the Porongos sequence occurred during the Neoproterozoic Brasiliano orogeny and comprises four ductile deformation phases (DB1–DB4), including two phases of isoclinal folding associated with shearing recorded in mylonitic layers, followed by closed NW-vergent folding and thrusting leading to formation of a thrust stack. Uplift of the basement and formation of late tectonic sedimentary basins occurred as a result of semi-ductile to brittle block faulting in a sinistral strike-slip regime. The Porongos sequence can be subdivided into a southeastern and a northwestern part. Trace element analyses as well as Sm–Nd and Rb–Sr geochemical data indicate partial melting and significant contamination by old continental crust for the metavolcanic rocks. The metavolcanic rocks show εNd(t=780 Ma) values of −20.64 and −21.72 (northwestern units) and −6.87 (southeastern sequence). The metasedimentary rocks were derived from late Palaeoproterozoic to Archaean sources, and the data indicate different sources for the northwestern and southeastern rock units of the Porongos sequence. εNd(t=780 Ma) are −6.25 and −6.85 in the southeastern units, with TDM model ages between 1734 and 1954 Ma, and vary between −14.72 and −17.96 in the northwestern parts, which have TDM model ages between 2346 and 2710 Ma. High 87Sr/86Sr(t) values between 0.7064 and 0.7286 confirm reworking of older crust. Isotopic signatures of the Porongos sequence do not show indications for a significant contribution from a Neoproterozoic juvenile source. A passive margin or continental rift environment is suggested for the tectonic setting of the Porongos belt, which is compatible with both deposition of shallow marine to deep marine sediments and stretching of continental crust leading to volcanism which is characterized by significant contamination by old continental crust.

Fracture paragenesis and microthermometry in Lisburne Group detachment folds: Implications for the thermal and structural evolution of the northeastern Brooks Range, Alaska

The distribution, character, and relative age of fractures in detachment folded Mississippian–Pennsylvanian Lisburne Group carbonates and overlying Permian–Triassic clastic rocks in the northeastern Brooks Range of northern Alaska provide important clues to the thermal and deformational sequence experienced by these rocks. Although paleothermal indices in the host rock limit the conditions of folding to temperatures equal to or less than 280C, field and petrographic relationships suggest that different fracture sets formed at different times during the deformational history of the rocks, providing a record of deformation under changing temperature and pressure conditions. These rocks probably initially entered the oil-generation window (80–140C) during the Early Cretaceous formation of the Colville basin via thrust loading by the Brooks Range to the south. Regional fractures formed during this time as a result of high pore pressures and low in-situ differential stresses. Shortening in these rocks related to the advancing northeastern Brooks Range fold and thrust belt began during the Late Cretaceous to early Tertiary. Early phases of detachment folding were via flexural slip, with associated fracturing. With continued shortening and growth of detachment folds, structural thickening resulted in deeper burial of the bottom part of the deforming wedge. Early fold-related fractures were subsequently overprinted by penetrative strain during peak folding at temperatures of approximately 280C. Continued shortening resulted in uplift and erosional unroofing at approximately 60 Ma. Late fold-related fractures formed at about 150C. Subsequent uplift of the thickened wedge through 60C occurred after about 25 Ma. Late pervasive extension fractures related to unroofing and/or regional stresses formed at relatively shallow depths and low temperatures, overprinting all the earlier fractures and penetrative structures.

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.

Morphogenesis of galls induced by Baccharopelma dracunculifoliae (Hemiptera: Psyllidae) on Baccharis dracunculifolia (Asteraceae) leaves

The commonest insect gall on Baccharis dracunculifolia (Asteraceae) leaves is induced by Baccharopelma dracunculifoliae (Hemiptera, Psyllidae). The gall-inducing insect attacks young leaves in both the unfolded and the fully expanded stages. Four developmental phases were observed in this type of gall: 1) A folding phase, during which the leaf lamina folded upward alongside the midrib and the edges of the upper portion of the leaf approached each other, forming a longitudinal slit. A single chamber was formed on the adaxial surface of the leaf; 2) A swelling phase, in which the folded leaf tissues thickened and the edges of the leaf drew closer together, narrowing the slit. In this phase the gall matured, turning succulent, fusiform and pale green. The single nymphal chamber was lined with white wax and was able to house from one to several nymphs; 3) A dehiscence phase, characterized by the opening of the slit to release inducers; and 4) A senescence phase, when the gall turned dark and dry. The dermal system of the mature gall was composed of a single-layered epidermis. The mesophyll was swollen, and the swelling was due mainly to hyperplasia of the parenchyma. The vascular tissues along the midrib vein were conspicuous and the perivascular fibers resembled parenchymal cells. The hypertrophied secretory cavities contained low lipophylic content. This gall does not form nutritive tissue, but salivary sheaths left by the inducers were observed near the parenchyma, vascular bundles and secretory cavities. This study complements our current knowledge of gall biology and sheds further light on the plasticity of plant tissues stimulated by biotic factors.

Geschwindigkeitsmessung der Stimmlippenbewegung

Introduction: Clinical examinations with short-interval, color-filtered double-flash stroboscopy enable us to estimate the movements of the vocal fold edges during phonation. So-called ‘displacement bands’ show the degree of the vibratory amplitude in these images. Using the two-point light projection method the displacement bands can be measured with very high accuracy. A combination of these two methods was used for velocity measurements of the vocal fold movements within the phonatory cycle. The aim of the study was to explore the influence of change in sound pressure level (SPL) and fundamental frequency (F₀) on the velocity of the horizontal amplitude of the vocal folds during phonation. Material and Methods: 40 healthy volunteers (31 women, 9 men, average age 24.8 years) were examined. During the special videostroboscopy with short-interval, color-filtered double flashes laser spots were projected onto the vocal folds by an endoscopic two-point light projection device. The subjects had to change their SPL and F₀ following a test protocol. During phonation they had to produce tones in low, middle and high chest voice as well as in falsetto register. Each subject was asked to do this in a soft, a modal, a loud and a very loud manner. Images of the phonatory cycle showing the vocal folds immmediately before collision were measured separately at three different positions. About 15,000 single measurements were evaluated. Results: The measured velocity of the horizontal amplitude during the closing phase of the vocal folds in chest voice was 30–160 cm/s. An increase in SPL resulted in an increasing velocity of the displacement bands. A change in F₀ did not lead to statistically relevant changes in the measured velocity. Conclusion: A combination of short-interval, color-filtered double-flash stroboscopy and a two-point light projection method enables measurements of the velocity of vocal fold movements. The SPL is the important factor for the velocity change within the phonatory cycle. F₀ seems not to covary with the velocity of the horizontal amplitude during the closing phase of the phonatory cycle.

Effect of Multiple Prolyl Isomerization Reactions on the Stability and Folding Kinetics of the Notch Ankyrin Domain: Experiment and Theory

Studies on the folding kinetics of the Notch ankyrin domain have demonstrated that the major refolding phase is slow, the minor refolding phase is limited by the isomerization of prolyl peptide bonds, and that unfolding is multiexponential. Here, we explore the relationship between prolyl isomerization and folding heterogeneity using a combination of experiment and simulation. Proline residues were replaced with alanine, both singly and in various combinations. These destabilizing substitutions combine to eliminate the minor refolding phase, although unfolding heterogeneity persists even when all seven proline residues are replaced. To test whether prolyl isomerization influences the major refolding phase, we modeled folding and prolyl isomerization as a system of sequential reactions. Simulations that use rate constants of the major folding phase of the Notch ankyrin domain to represent intrinsic folding indicate that even with seven prolyl isomerization reactions, only two significant phases should be observed, and that the fast observed phase provides a good approximation of the intrinsic folding in the absence of prolyl isomerization. These results indicate that the major refolding phase of the Notch ankyrin domain reflects an intrinsically slow folding transition, rather than coupling of fast folding events with slow prolyl isomerization steps. This is consistent with the observation that the single observed refolding phase of a construct in which all proline residues are replaced remains slow. Finally, the simulation fails to produce a second unfolding phase at high urea concentrations, indicating that prolyl isomerization does not play a role in the three-state mechanism that leads to this heterogeneity.