Template-constrained somatostatin analogues: a biphenyl linker induces a type-V' turn.
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Biphenyl Compounds↗
Biomedical subjects
H Roder
Publications and source records attributed to H Roder.
Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate.
Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.
Bacterial Proteins↗
Structural and kinetic characterization of early folding events in beta-lactoglobulin.
We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.
Animals↗
Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy.
We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.
Amino Acid Sequence↗
Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics.
The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.
Amides↗
The evolutionarily conserved Dim1 protein defines a novel branch of the thioredoxin fold superfamily.
Dim1 is a small evolutionarily conserved protein essential for G2/M transition that has recently been implicated as a component of the mRNA splicing machinery. To date, the mechanism of Dim1 function remains poorly defined, in part because of the absence of informative sequence homologies between Dim1 and other functionally defined proteins or protein domains. We have used a combination of molecular modeling and NMR structural analysis to demonstrate that approximately 125 of the 142 amino acids of human Dim1 (hDim1) define a novel branch of the thioredoxin fold superfamily. Mutational analysis of Dim1 based on the predicted fold indicates that alterations in the region corresponding to the thioredoxin active site do not affect Dim1 activity. However, removal of a very short carboxy-terminal extension generates a dominant negative form of the protein [hDim1-(1-128)] that when overproduced induces cell cycle arrest in G2, via a mechanism likely to involve alteration of Dim1 association with partner molecules. In sum, this study identifies the Dim1 proteins as a novel sixth branch of the thioredoxin superfamily involved in cell cycle.
Amino Acid Sequence↗
Activation of neuronal extracellular receptor kinase (ERK) in Alzheimer disease links oxidative stress to abnormal phosphorylation.
Responses to increased oxidative stress may be the common mechanism responsible for the varied cytopathology of Alzheimer disease (AD). A possible link in support of this hypothesis is that one of the most striking features of AD, the abnormal accumulation of highly phosphorylated tau and neurofilament proteins, may be brought about by extracellular receptor kinase (ERK) whose activation is a common response to oxidative stress. In this study, we demonstrate that activated ERK is specifically increased in the same vulnerable neurons in AD that are the site of oxidative damage and abnormal phosphorylation. These findings suggest that ERK dysregulation, likley resulting from oxidative stress, could play an important role in the increased phosphorylation of cytoskeletal proteins observed in AD.
Adult↗
Solution structure and dynamics of a de novo designed three-helix bundle protein.
Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, alpha3D, is reported. The structure of alpha3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.
Amino Acid Sequence↗
Methods for exploring early events in protein folding.
Progress in understanding dynamic aspects of protein folding relies on the continuing development of methods for obtaining more detailed structural information on the transient conformational ensembles that often appear within microseconds of initiating refolding. Advances in rapid mixing and other time-resolved spectroscopic methods have made it possible to explore some of the earliest stages of folding, including the initial formation of compact states, which is determined by the presence of a sequence-specific kinetic barrier, as well as the 'downhill' folding kinetics after the rate-limiting barrier has been crossed.
Circular Dichroism↗
Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing.
For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600-700 micros), which is followed by a slower phase with a denaturant-dependent time constant (2-30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U right harpoon over left harpoon I right harpoon over left harpoon N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.
Fluorescence↗
Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state.
Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.
Animals↗
Definition of amide protection factors for early kinetic intermediates in protein folding.
Hydrogen-deuterium exchange experiments have been used previously to investigate the structures of well defined states of a given protein. These include the native state, the unfolded state, and any intermediates that can be stably populated at equilibrium. More recently, the hydrogen-deuterium exchange technique has been applied in kinetic labeling experiments to probe the structures of transiently formed intermediates on the kinetic folding pathway of a given protein. From these equilibrium and nonequilibrium studies, protection factors are usually obtained. These protection factors are defined as the ratio of the rate of exchange of a given backbone amide when it is in a fully solvent-exposed state (usually obtained from model peptides) to the rate of exchange of that amide in some state of the protein or in some intermediate on the folding pathway of the protein. This definition is straightforward for the case of equilibrium studies; however, it is less clear-cut for the case of transient kinetic intermediates. To clarify the concept for the case of burst-phase intermediates, we have introduced and mathematically defined two different types of protection factors: one is P struc, which is more related to the structure of the intermediate, and the other is P app, which is more related to the stability of the intermediate. Kinetic hydrogen-deuterium exchange data from disulfide-intact ribonuclease A and from cytochrome c are discussed to explain the use and implications of these two definitions.
Amides↗
Determinants of protein hydrogen exchange studied in equine cytochrome c.
The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 10(3) are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (OH(-)-ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 angstroms. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.
Animals↗
A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.
A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 micros of its initiation and that the dead time of the measurement is 45 +/- 5 micros, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from approximately 45 micros to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.
Biophysics↗
Changes in dendritic structure and function following hippocampal lesions: correlations with developmental events?
Recovery after nervous system lesions may lead to partial re-institution of developmental schemes and processes. Here we review several of these proposed schemes, with the conclusion that though some processes may involve re-expression of embryonic phenotypes, there are many processes invoked during recovery from lesions that do not mirror developmental phenomena. The inability to fully revert to embryonic schemes because of adult phenotype may partially account for the decreased recovery observed in adults compared to that noted after lesions during development.
Animals↗
Amide protection in an early folding intermediate of cytochrome c.
BACKGROUND: For many proteins, compact states appear long before the rate-limiting step in the formation of the native structure. A key issue is whether the initial collapse of the chain is driven by random or more specific hydrophobic interactions. RESULTS: Hydrogen-exchange labeling coupled with NMR was used to monitor the formation of stable hydrogen-bonded and solvent-excluded structure in horse cytochrome c (cyt c). Protection was measured using a hydrogen exchange/folding competition protocol at variable pH and short competition time (2 ms). Protection factors of threefold to eightfold were observed in all three alpha helices of cyt c, whereas other regions showed no significant protection. This suggests that the compact states that are present contain segments of marginally stable hydrogen-bonded structure. When the intermediate(s) are destabilized, only amide protons from Cys14, Ala15 and His18 show significant protection, indicating a region of persistent residual structure near the covalently bound heme group in the unfolded protein. Fluorescence-detected stopped-flow studies showed that the maximum protection factor in the early intermediate is consistent with its unfolding equilibrium constant. CONCLUSIONS: Together with previous fluorescence and CD results, the observed pattern of amide protection is consistent with the early formation of an alpha-helical core domain in an ensemble of compact states, indicating that efficient folding is facilitated by stepwise acquisition of native structural elements. These specific early interactions are established on the sub-millisecond time scale, prior to the rate-limiting step for folding.
Amides↗
Evidence for barrier-limited protein folding kinetics on the microsecond time scale.
Although important structural events in protein folding are known to occur on the submillisecond time scale, the limited time resolution of conventional kinetic methods has precluded direct observation of the initial collapse of the polypeptide chain. A continuous-flow capillary mixing method recently developed by us made it possible to account for the entire fluorescence change associated with refolding of cytochrome c from approximately 5-10(-5)-10(2) s, including the previously unresolved quenching of Trp 59 fluorescence (burst phase) indicative of the formation of compact states. The kinetics of folding exhibits a major exponential process with a time constant of approximately 50 micros, independent of initial conditions and heme ligation state, indicating that a common free energy barrier is encountered during the initial collapse of the polypeptide chain. The resulting loosely packed intermediate accumulates prior to the rate-limiting formation of specific tertiary interactions, confirming previous indications that folding involves at least two distinct stages.
Animals↗
[Expression of mutant horse cytochrome c genes in Escherichia coli].
Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants. These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome. Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture. This is by an order of magnitude greater than the expression previously achieved in yeast. A series of horse cytochrome c mutants were obtained in this way.
Animals↗