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Understanding how proteins adopt their unique native structures requires a complete structural characterization of the rate-limiting transition state(s) along the folding pathway. By definition, transition states are not significantly populated and are only accessible via folding kinetics studies. In this respect, interpreting the kinetic effects of amino acid substitutions (especially to Ala) via Φ-value analysis is the most common method to probe the structure of these transient, yet important states. A critical review of the key assumptions required for rigorous interpretation of Φ values reveals that a multiple substitution strategy in which a position of interest is mutated to a variety of amino acids, and not exclusively to Ala, provides the best means to characterize folding transition states. This approach has proven useful in revealing nonnative interactions and (or) conformations in folding transition states. Moreover, by simultaneously examining the folding kinetics of multiple substitutions made at a single surface-exposed position using the Bronsted analysis the backbone conformation in a folding transition state can be investigated. For folding equilibria with exchange rates on the order of milliseconds, the kinetic parameters for Φ-value analysis can be obtained from NMR relaxation dispersion experiments, under fully native conditions, along with a wealth of high-resolution structural information about the states in exchange (native, denatured, and intermediate states that populate the pathway). This additional structural information, which is not readily obtained through stopped-flow based methods, can significantly facilitate the interpretation of Φ values because it often reports on the validity of the assumptions required for a rigorous interpretation of Φ values. Key words: protein folding, Φ-value analysis, SH3 domains, mutagenesis, folding transition states. La comprehension du mecanisme par lequel les proteines adoptent leurs structures natives uniques requiert une caracterisation structurale complete de l'etat ou des etats de transitions limitants durant le processus de repliement. Par definition, les etats de transition ne sont pas significativement abondants et ne sont accessibles que par l'intermediaire d'etudes cinetiques de repliement. A cet effet, l'interpretation des effets cinetiques de la substitution d'acides amines (specialement par une Ala) a l'aide d'une analyse des valeurs de Φ est la methode la plus utilisee pour sonder ces etats transitoires mais importants. Une revue critique des postulats de base requis pour interpreter rigoureusement les valeurs de Φ revele qu'une strategie utilisant de multiples substitutions, par laquelle un acide amine situe dans une position d'interet est mute en une variete d'acides amines et non seulement en Ala, fournit le meilleur moyen de caracteriser les etats de transition de repliement. Cette approche a demontre son utilite en revelant les interactions/conformations non-natives dans les etats de transition de repliement. De plus, en examinant simultanement des cinetiques de repliement de multiples substitutions realisees sur un site unique expose a la surface par une analyse de Bronsted, la conformation d'une chaine dans un etat de transition de repliement donne peut etre etudiee. Dans le contexte d'un repliement a l'equilibre avec des taux d'echange de l'ordre de la milliseconde, les parametres cinetiques de l'analyse des valeurs de Φ peuvent etre obtenus par des experiences en RMN de relaxation/dispersion, sous des conditions pleinement natives, avec une abondance d'informations structurales a haute resolution des etats en echange (natif, denature et etats intermediaires qui jalonnent ce sentier). Cette information structurale additionnelle, qui n'est pas facilement obtenue par des methodes en stop-flow, peut faciliter de facon significative l'interpretation des valeurs de Φ car elle temoigne de la validite des postulats requis pour interpreter rigoureusement les valeurs de Φ. Mots-cles: repliement des proteines, analyse des valeurs de Φ, domaines SH3, mutagenese, etats de transition de repliement. [Traduit par la Redaction], Introduction to Φ-value analysis In the quest to understand the biophysical principles underlying the relationship between the amino acid sequence of a protein and its three-dimensional structure, many studies have [...]

New labeling approach and NMR experiments are described along with those that has probed the exchange by using [super 15]N and [super 1]H N nuclei. The experimental data has showed that for many systems accurate excited-state chemical shifts can be measured, thus opening a possibility of obtaining quantitative structural information of invisible states of the sort that is recorded on ground states of proteins.

Experimental studies of protein folding frequently are consistent with two-state folding kinetics. However, recent NMR relaxation dispersion studies of several fast-folding mutants of the Fyn Src homology 3 (SH3) domain have established that folding proceeds through a low-populated on-pathway intermediate, which could not be detected with stopped-flow experiments. The dispersion experiments provide precise kinetic and thermodynamic parameters that describe the folding pathway, along with a detailed site-specific structural characterization of both the intermediate and unfolded states from the NMR chemical shifts that are extracted. Here we describe NMR relaxation dispersion [PHI]-value analysis of the A39V/N53P/V55L Fyn SH3 domain, where the effects of suitable point mutations on the energy landscape are quantified, providing additional insight into the structure of the folding intermediate along with per-residue structural information of both rate-limiting transition states that was not available from previous studies. In addition to the advantage of delineating the full three-state folding pathway, the use of NMR relaxation dispersion as opposed to stopped-flow kinetics to quantify [PHI] values facilitates their interpretation because the obtained chemical shifts monitor any potential structural changes along the folding pathway that might be introduced by mutation, a significant concern in their analysis. [PHI]-Value analysis of several point mutations of A39V/N53P/V55L Fyn SH3 establishes that the [[beta].sub.3]-[[beta].sub.4]-hairpin already is formed in the first transition state, whereas strand [[beta].sub.1], which forms nonnative interactions in the intermediate, does not fully adopt its native conformation until after the final transition state. The results further support the notion that on-pathway intermediates can be stabilized by nonnative contacts. chemical exchange | Carr-Purcell-Meiboom-Gill | Fyn SH3 | protein folding intermediate | transition state

Various studies are conducted to explain the structural properties, as well as the dynamics of the stable nonfibrillar oligomers that are formed by the human prion protein (PrP) residues. These oligomers are shown to be highly associated with various neurodegenerative diseases.

The article discusses a new approach, which can be used for the analysis of the various isoleucine side-chain conformations taking place in the ground and the excited states of proteins from chemical shifts. The various benefits and applications of the methodology are also explained.

The article explains a simple approach, which is used for the extraction of the Leu side-chain conformations taking place in the excited protein states. The methodology is shown to be extremely useful for the characterization of the structural, as well as the dynamic properties of the different states.

A simple experiment to obtain the sign of [super 1]H[alpha] [Delta] values by measuring off-resonance [super 1]H[alpha] decay rates, [R.sub.1[rho]] and using weak proton spin-lock fields is presented. The experiment provides possible route to determine [super 1]H[alpha] chemical shifts of the invisible excited state which could be used as powerful restraints in defining the structural properties of such elusive conformers.

The study results of [15.sup.N] relaxation dispersion of protein folding for a pair of SH3 domains, including the G48V mutant of a stabilized SH3 module from Abp1p and the A39V/N53P/V55L mutant of an SH3 domain from Fyn is presented. The results from the study suggest that formation of the intermediate state observed originally in studies of G48V and G48M Fyn SH3 might be a general feature of SH3 domain folding.

The three-site exchange folding reaction of an (super 15)N-labeled, Gly48Met mutant of the Fyn SH3 domain is characterized by using a suit of six dispersion experiment that measures exchange contribution to backbone (super 1)H and (super 15)N traverse relaxation rates in proteins. The experiments facilitate the complete reconstruction of (super 1)H and (super 15)N correlation spectra for the unfolded and intermediate states that are invisible in even the most sensitive of NMR experiments.

Purpose: To characterize the proton exchange in aqueous urea solutions using a modified version of the WEX II filter at high magnetic field, and to assess the feasibility of performing quantitative urea CEST MRI on a 3T clinical MR system. Methods: In order to study the dependence of the exchange‐rate constant ksw of urea as a function of pH and T, the WEX‐spectra were acquired at 600 MHz from urea solutions in a pH range from 6.4 to 8.0 and a temperature range from T=22∘C to 37∘C. The CEST experiments were performed on a 3T MRI scanner by applying a train of 50 Gaussian‐shaped pulses, each 100‐millisecond long with a spacing of 100 milliseconds, for saturation. Exchange rates of urea were calculated using the (extended) AREX metric. Results: The results showed that proton exchange in aqueous urea solutions is acid and base catalyzed with the rate constants: ka=(9.95±1.1)×106 l/(mol·s) and kb=(6.21±0.21)×106 l/(mol·s), respectively. Since the urea protons undergo a slow exchange with water protons, the CEST effect of urea can be observed efficiently at 3T. However, in neutral solutions the exchange rate of urea is minimal and cannot be estimated using the quantitative CEST approach. Conclusions: By means of the WEX‐spectroscopy, the kinetic parameters of the proton exchange in urea solutions have been determined. It was also possible to estimate the exchange rates of urea in a broad range of pH values using the CEST method at a clinical scanner. [ABSTRACT FROM AUTHOR]

Deoxyribose-5-phosphate aldolase (DERA) catalyses the reversible conversion of 2-deoxyribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde. For industrial applications, this enzyme is used in organic synthesis for aldol reactions between acetaldehyde as a donor and a wide range of aldehydes as acceptors. Here, we present a near complete set of sequence-specific H, C and N resonance assignments of a 28 kDa monomeric variant of the Escherichia coli DERA. These assignments provide the basis for ongoing structural and dynamic analysis of DERA substrate specificity. [ABSTRACT FROM AUTHOR]

The third WW domain ( WW3*) of the ubiquitin ligase human neuronal precursor cell expressed developmentally downregulated gene 4-1 ( hNedd4-1) was reported to bind its PY motif peptide by a coupled folding-binding equilibrium. However, it is unknown whether these thermodynamic properties are retained in the context of neighboring hNedd4-1 domains. In this report, NMR data show that the WW3* displays a fold-unfold equilibrium in the presence of neighboring WW domains, and that similar fold-unfold equilibria also likely exist for neighboring WW domains. These equilibria are quenched upon interaction with peptide. Thus, the binding mechanism of hNedd4-1 WW domains to proteins involves coupled folding and binding equilibria, and this mechanism may be a general feature that modulates peptide affinities of WW domains. [ABSTRACT FROM AUTHOR]

The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na+ channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the a-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the 15N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. ¹HN and 15N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ~20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium. [ABSTRACT FROM AUTHOR]

A hallmark of Alzheimer’s disease (AD) is the accumulation of extracellular amyloid-β (Aβ) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Aβ (pEAβ) has been described as a major compound of Aβ species in senile plaques. pEAβ is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and β-sheet stabilization compared to non-modified Aβ. Here we characterized recombinant pEAβ(3–40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Aβ(1–40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAβ(3–40) shows an increased tendency to form β-sheet-rich structures in 20% TFE containing solutions where Aβ(1–40) forms α-helices. Aggregation kinetics of pEAβ(3–40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to Aβ(1–40), which lacks ThT positive structures under the same conditions. Transmission electron microscopy confirms that pEAβ(3–40) aggregated to large fibrils and high molecular weight aggregates in spite of the presence of the helix stabilizing co-solvent TFE. High resolution NMR spectroscopy of recombinantly produced and uniformly isotope labeled [U-15N]-pEAβ(3–40) in TFE containing solutions indicates that the pyroglutamate formation affects significantly the N-terminal region, which in turn leads to decreased monomer stability and increased aggregation propensity. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ and comparison to its non-modified variant has so far been seriously hampered by the lack of highly pure recombinant and isotope-enriched protein. Here we present, to our knowledge, for the first time a reproducible protocol for the production of pEAβ from a recombinant precursor expressed in E. coli in natural isotope abundance as well as in uniformly [U-15N]- or [U-13C, 15N]-labeled form, with yields of up to 15 mg/l E. coli culture broth. The chemical state of the purified protein was evaluated by RP-HPLC and formation of pyroglutamate was verified by mass spectroscopy. The recombinant pyroglutamate-modified Aβ peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEAβ40 and pEAβ42 allowed us to perform heteronuclear multidimensional NMR spectroscopy in solution and to sequence-specifically assign the backbone resonances under near-physiological conditions. Our results suggest that the presented method will be useful in obtaining cost-effective high-quality recombinant pEAβ40 and pEAβ42 for further physiological and biochemical studies. [ABSTRACT FROM AUTHOR]

The formation of nonfibrillar oligomers has been proposed as a common element of the aggregation pathway of proteins and peptides associated with neurodegenerative diseases such as Alzheimer's and Creutzfeldt-Jakob disease. While fibrillar structures have long been considered indicators of diseases linked with the accumulation of amyloid plaques, it has more recently been proposed that amyloid oligomers are in fact the cytotoxic form. Here we describe the local structure and dynamics of stable oligomers formed by a peptide comprising residues 106-126 of the human prion protein (PrP). Structural constraints from solid-state NMR reveal quaternary packing interactions within the hydrophobic core, similar to those previously reported for amyloid fibrils formed by this peptide, and consistent with structural studies of oligomers formed by the Alzheimer's 13-amyloid peptide. However, a hydration-dependent increase in disorder is observed for nonfibrillar oligomers of PrP(106-126). In solution NMR spectra we observe narrow 1H and 13C resonances corresponding to a monomer in exchange with the `∼30 nm diameter nonfibrillar oligomers, giving additional information on the molecular structure of these species. Taken together, our data support a model in which the local structure of the oligomers contains the basic elements of amyloid fibrils, but with long-range disorder and local mobility that distinguishes these assemblies from the fibrillar form of PrP(106-126). These characteristics may provide a basis for the differing biological activities of amyloid fibrils and oligomers. [ABSTRACT FROM AUTHOR]

NMR relaxation dispersion spectroscopy is a powerful method for studying protein conformational dynamics whereby visible, ground and invisible, excited conformers interconvert on the millisecond time-scale. In addition to providing kinetics and thermodynamics parameters of the exchange process, the CPMG dispersion experiment also allows extraction of the absolute values of the chemical shift differences between interconverting states, $$ \left| {\Updelta \tilde{\omega }} \right| $$, opening the way for structure determination of excited state conformers. Central to the goal of structural analysis is the availability of the chemical shifts of the excited state that can only be obtained once the signs of $$ \Updelta \tilde{\omega } $$ are known. Herein we describe a very simple method for determining the signs of 1HN $$ \Updelta \tilde{\omega } $$ values based on a comparison of peak positions in the directly detected dimensions of a pair of 1HN–15N correlation maps recorded at different static magnetic fields. The utility of the approach is demonstrated for three proteins that undergo millisecond time-scale conformational rearrangements. Although the method provides fewer signs than previously published techniques it does have a number of strengths: (1) Data sets needed for analysis are typically available from other experiments, such as those required for measuring signs of 15N $$ \Updelta \tilde{\omega } $$ values, thus requiring no additional experimental time, (2) acquisition times in the critical detection dimension can be as long as necessary and (3) the signs obtained can be used to cross-validate those from other approaches. [ABSTRACT FROM AUTHOR]

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible ‘excited’ conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Δϖ|), that inform on the structural properties of the excited state(s). The sign of Δϖ is, however, not available from CPMG data. Here we present one-dimensional NMR experiments for measuring the signs of 1HN and 13Cα Δϖ values using weak off-resonance R1 ρ relaxation measurements, extending the spin-lock approach beyond previous applications focusing on the signs of 15N and 1Hα shift differences. The accuracy of the method is established by using an exchanging system where the invisible, excited state can be converted to the visible, ground state by altering conditions so that the signs of Δϖ values obtained from the spin-lock approach can be validated with those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring the signs of chemical shift differences. For the Abp1p and Fyn SH3 domains considered here it is found that while H(S/M)QC measurements provide signs for more residues than the spin-lock data, the two different methodologies are complementary, so that combining both approaches frequently produces signs for more residues than when the H(S/M)QC method is used alone. [ABSTRACT FROM AUTHOR]

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes. [ABSTRACT FROM AUTHOR]

Abstract: Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central β-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal β-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F↔I↔U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (<25 °C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F↔I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data. [Copyright &y& Elsevier]

Birch pollinosis is one of the prevailing allergic diseases. In all, 5–20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca2+-binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal α-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca2+ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4. [Copyright &y& Elsevier]

Studies the solution structure of human proguanylin, a 94-amino acid prohormone. Structure determination; Molecular dynamics; Spectrum of proguanylin.

The article presents a study on the value measurements of 1Hα chemical shift differences between protein and ground states with the use of nuclear magnetic resonance (NMR) spectroscopy. It notes that ground and protein states are formed during the exchange process which can be of significance to the measurement of chemical shift values. It also concludes that conformational ensembles can be defined by the shifts in the biological function.

Presents a letter to the editor focusing on experiments done on allergenic 2S albumins from peanut.