Role of visual evoked potentials in the assessment and management of optic pathway gliomas in children.

OBJECTIVE: The aim of this study is to investigate the role of pattern reversal visual evoked potentials (pVEPs) in the screening and monitoring of optic pathway gliomas (OPGs) in children with and without neurofibromatosis type 1. METHODS: A review of the English literature published between 1980 and 2012 was performed, with comparison of results of retro- and prospective studies. RESULTS: Pattern reversal VEPs have a high sensitivity (85.7-100 %) for the diagnosis of OPGs, moreover they are safe and cost-effective. Conversely, they have a low specificity (43-83 %) and are not widely available. Besides, pattern reversal VEP results can be unreliable in young children, because of the need for a good cooperation. The studies that were analyzed have drawbacks, including the small sample size, the retrospective design, the differences in gold standard for diagnosis, the different interpretation of small changes in VEP results and the lack of control groups. CONCLUSION: There is still debate about the gold standard for the screening and follow-up of OPGs. The added value of pVEPs to the ophthalmic examination is controversial. Randomized controlled trials or prospective multicentre studies are necessary to assess with sufficient accuracy the sensitivity and specificity of pattern reversal VEPs in the screening for OPGs and its follow-up.

Structural characterisation of apoflavodoxin shows that the location of the stable nucleus differs among proteins with a flavodoxin-like topology.

The structural characteristics of Azotobacter vinelandii apoflavodoxin II have been determined using multidimensional NMR spectroscopy. Apoflavodoxin has a stable, well-ordered core but its flavin binding region is flexible. The local stability of apoflavodoxin was probed using hydrogen/deuterium exchange measurements. The existence of an apoflavodoxin equilibrium folding intermediate is inferred from the non-coincidence of CD and fluorescence unfolding curves obtained for the guanidinium hydrochloride induced unfolding of apoflavodoxin. We suggest that the structured part of the putative intermediate is composed of the elements of secondary structure which have the slowest exchanging amide protons in the native protein. These elements are strands beta1, beta3, beta4 and beta5a and helices alpha4 and alpha5. We propose that it is a general feature of flavodoxins that the stable nucleus resides in the C-terminal part of these proteins. The results on flavodoxin are compared with those on two sequentially unrelated proteins sharing the flavodoxin-like fold: Che Y and cutinase. It is shown that the stable nucleus is found in different parts of the flavodoxin-like topology.

Local structure due to an aromatic-amide interaction observed by 1H-nuclear magnetic resonance spectroscopy in peptides related to the N terminus of bovine pancreatic trypsin inhibitor.

A synthetic peptide corresponding to the 15 N-terminal residues of bovine pancreatic trypsin inhibitor, with serine replacing the two cysteine residues, has been characterized by 1H-nuclear magnetic resonance spectroscopy. This peptide has a very disordered conformation that is essentially the same when it is part of the analogue of the (30-51) one-disulphide intermediate in folding. This confirms the conclusion of a previous paper, that the (30-51) intermediate is partially folded, with the N-terminal segment disordered. Local elements of non-random conformation were observed in the peptide. Especially prominent was an apparently electrostatic interaction between the face of the aromatic ring of Tyr10 and the amide group of Gly12, which caused the latter to have a very anomalous chemical shift. A similar interaction was observed in shorter peptides, especially in tetrapeptides with the sequences Tyr/Phe-X-Gly-Y. The local nature of this interaction indicates that it should be a general feature in peptides and in unfolded proteins with such a sequence.

Partially folded conformation of the (30-51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. 1H and 15N resonance assignments and determination of backbone dynamics from 15N relaxation measurements.

An analogue of the important folding intermediate of BPTI with only the disulphide bond between Cys30 and Cys51 has been characterized by 1H and 15N NMR techniques. In particular, the dynamics of the polypeptide backbone were characterized using (1H)-15N NOE and 15N T1 and T2 relaxation data. The intermediate is partially folded, with part of the polypeptide chain stably folded and the remainder flexible or unfolded. The folded portion consists of the major elements of native-like secondary structure interacting through the hydrophobic core of the molecule. The 15N relaxation data show that the N-terminal 15 residues are very flexible, and the (1H, 1H) NOESY data show that these residues have no NOE interactions with the remainder of the molecule. The segment of residues 37 to 41 is also flexible. These observations explain why during folding this intermediate most readily forms any of the possible disulphide bonds between Cys5, Cys14 and Cys38, including the non-native 5-14 and 5-38 bonds. The native-like folded portion of the molecule limits the possible disulphide bonds that can be formed to those in the remainder of the polypeptide chain. Also, forming the non-native disulphide bonds need not involve any disruption of that folded structure, as the Cys residues involved are in flexible regions of the molecule.

(14-38, 30-51) double-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor: a two-dimensional 1H nuclear magnetic resonance study.

An analogue of the BPTI folding intermediate that contains only the disulphide bonds between Cys14 and Cys38 and between Cys30 and Cys51 has been prepared in Escherichia coli by protein engineering methods. The other two Cys residues of native BPTI (at positions 5 and 55) have been replaced by Ser. Essentially complete proton resonance assignments of the analogue were obtained by employing two-dimensional 1H nuclear magnetic resonance techniques. The intermediate has a more extended conformation in the N-terminal (residues 1 to 7) region and there are other differences in the C-terminal (residues 55 to 58) region. The remainder of the protein is substantially identical to native BPTI. The conformational properties of the analogue can explain several aspects of the kinetic role that the normal (14-38, 30-51) intermediate plays in the folding of BPTI.

Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor.

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.

Kinetic roles and conformational properties of the non-native two-disulphide intermediates in the refolding of bovine pancreatic trypsin inhibitor.

The most productive folding pathway of reduced bovine pancreatic trypsin inhibitor (BPTI) proceeds through the disulphide intermediates (30-51), (30-51, 5-14), and (30-51, 5-38); these are important kinetic intermediates in folding, even though the latter pair contain non-native disulphide bonds. Analogues of these intermediates have been prepared by protein engineering methods and their conformational properties examined by circular dichroism and 1H-nuclear magnetic resonance. The (30-51), (30-51, 5-14) and (30-51, 5-38) analogues exhibit comparable degrees of stable structure, which cannot include those portions of the polypeptide chain involving Cys5, Cys14 and Cys38. These properties are consistent with the roles of (30-51, 5-14) and (30-51, 5-38) in the folding pathway of BPTI, which demand that they exhibit a considerable degree of conformational flexibility in part of the molecule.

1H NMR analysis of the partly-folded non-native two-disulphide intermediates (30-51,5-14) and (30-51,5-38) in the folding pathway of bovine pancreatic trypsin inhibitor.

The conformational properties of analogues of the (30-51,5-14) and (30-51,5-38) disulphide intermediates in refolding of reduced BPTI, with non-native second disulphide bonds, have been characterized in detail by 1H NMR analysis. They are shown to have partly-folded conformations, very similar to that of the (30-51) one-disulphide intermediate from which they arise during folding. The non-native disulphide bonds are formed in flexible or unfolded parts of the polypeptide chain; they do not disrupt the folded portion nor do they introduce substantial non-native conformation. The conformational properties of these intermediates explain their important roles in the folding pathway.

Apoflavodoxin (un)folding followed at the residue level by NMR.

The denaturant-induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H-15N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H-15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.

The 5-55 single-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor.

An analogue of the BPT1 folding intermediate that contains only the disulphide bond between Cys-5 and Cys-55 has been prepared by mutation of the other four Cys residues to Ser. On the basis of its circular dichroism and 1H-nuclear magnetic resonance spectra and its electrophoretic mobility, this intermediate is shown to be at least partially folded at low temperatures. This probably accounts for several of the unique properties of this intermediate observed during folding.

Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story.

In this review, the experimental results obtained on the folding and stability of Azotobacter vinelandii flavodoxin are summarised. By doing so, three main spectroscopic techniques used to investigate protein folding and stability are briefly introduced. These techniques are: circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy in combination with the hydrogen exchange methodology. Results on the denaturant-induced and thermal equilibrium unfolding of apoflavodoxin from A. vinelandii, i.e. flavodoxin in the absence of the riboflavin-5'-monophosphate (FMN) cofactor, are discussed. A scheme for the equilibrium unfolding of apoflavodoxin is presented which involves a relatively stable molten globule-like intermediate. Denaturant-induced apoflavodoxin (un)folding as followed at the residue-level by NMR shows that the transition of native A. vinelandii apoflavodoxin to its molten globule state is highly co-operative. However, the unfolding of the molten globule to the unfolded state of the protein is non-co-operative. A comparison of the folding of A. vinelandii flavodoxin with the folding of flavodoxin from Anaboena PCC 7119 is made. The local stabilities of apo- and holoflavodoxin from A. vinelandii as measured by NMR spectroscopy are compared. Both Che Y and cutinase, which have no sequence homology with apoflavodoxin but which share the flavodoxin-like topology, have stabilisation centres different from that of apoflavodoxin from A. vinelandii. The stable centres of structurally similar proteins can thus reside in different parts of the same protein topology. Insight in the variations in (local) unfolding processes of structurally similar proteins can be used to stabilise proteins with a flavodoxin-like fold. Finally, the importance of some recent experimental and theoretical developments for the study of flavodoxin folding is briefly discussed.

5-fluorotryptophan as dual probe for ground-state heterogeneity and excited-state dynamics in apoflavodoxin.

The apoflavodoxin protein from Azotobacter vinelandii harboring three tryptophan (Trp) residues, was biosynthetically labeled with 5-fluorotryptophan (5-FTrp). 5-FTrp has the advantage that chemical differences in its microenvironment can be sensitively visualized via (19)F NMR. Moreover, it shows simpler fluorescence decay kinetics. The occurrence of FRET was earlier observed via the fluorescence anisotropy decay of WT apoflavodoxin and the anisotropy decay parameters are in excellent agreement with distances between and relative orientations of all Trp residues. The anisotropy decay in 5-FTrp apoflavodoxin demonstrates that the distances and orientations are identical for this protein. This work demonstrates the added value of replacing Trp by 5-FTrp to study structural features of proteins via (19)F NMR and fluorescence spectroscopy.

Doubly sensitivity-enhanced 3D TOCSY-HSQC.

Recently, strategies for double sensitivity enhancement in heteronuclear three-dimensional NMR experiments were introduced (Krishnamurthy, V.V. (1995) J. Magn. Reson., B106, 170-177; Sattler et al. (1995) J. Biomol. NMR, 6, 11-22; Sattler et al. (1995) J. Magn. Reson., B108, 235-242). Since a sensitivity enhancement of a factor 2(1/2) can be achieved for each indirect dimension, nD spectra can theoretically be enhanced up to a factor of 2(((n-1)/2)). We propose and analyze a doubly enhanced three-dimensional TOCSY-HSQC sequence. The application of the doubly enhanced three-dimensional {(15)N, (1)H} TOCSY-HSQC sequence is shown for uniformly (13)C-/(15)N- and (15)N-labeled samples of the relatively large Azotobacter vinelandii flavodoxin II (179 amino acids). The main factors that contribute to the final signal-to-noise enhancement have been systematically investigated. The sensitivity enhancement obtained for the doubly enhanced TOCSY-HSQC pulse sequence as compared to the standard (unenhanced) version is close to the theoretically expected factor of two.

Secondary and tertiary structure characteristics of Megasphaera elsdenii flavodoxin in the reduced state as determined by two-dimensional 1H NMR.

The secondary structure of two-electron-reduced Megasphaera elsdenii flavodoxin has been determined by visual, qualitative inspection of the sequential connectivities involving C alpha H, C beta H and NH protons observed in NOESY (two-dimensional nuclear Overhauser enhancement spectroscopy) spectra. Results from an amide proton exchange experiment were used to confirm the secondary structure assignment and to demonstrate the compactness and stability of the protein. After the secondary structure elements were established, the global fold of the protein and the flavin binding site have been determined using nonsequential interresidual NOE connectivities as primary source of information. The secondary structure and the global fold of M. elsdenii and Clostridium MP flavodoxin appeared to be very similar, differences are observed however. M. elsdenii flavodoxin consists of a central parallel beta-sheet including five strands surrounded on both sides by a pair of alpha-helices.

Three-dimensional correlated NMR study of Megasphaera elsdenii flavodoxin in the oxidized state.

The value of a three-dimensional (3D) non-selective total correlation/nuclear Overhauser enhancement spectroscopy (TOCSY-NOESY) spectrum for making sequential resonance assignments in proteins is demonstrated using the relatively large Megasphaera elsdenii flavodoxin (molecular mass 15 kDa) in the oxidized state. An easy and concise method for the analysis of 3D-NMR spectra and a strategy for the resonance assignment of 3D-NMR protein spectra is introduced. In this context, non-selective TOCSY-NOESY is compared with selective TOCSY-NOESY and non-selective NOESY-TOCSY. Sequential assignments in various secondary structure elements of flavodoxin are made using the method of analysis introduced. NOEs not previously identified in 2D-NMR spectra due to resonance overlap are found in the 3D Clean-TOCSY-NOESY spectrum. Also additional side-chain assignments could be made.

The partially folded conformation of the Cys-30 Cys-51 intermediate in the disulfide folding pathway of bovine pancreatic trypsin inhibitor.

The best-characterized protein folding pathway is that of bovine pancreatic trypsin inhibitor, which folds from the reduced form through a series of disulfide bond intermediates. The crucial one-disulfide intermediate of bovine pancreatic trypsin inhibitor with the disulfide bond between Cys-30 and Cys-51 is shown here to have a partially folded conformation in which the major elements of secondary structure interact via a core of apolar side chains, which resembles part of the native conformation. The stability of this structure can account for the predominance of this one-disulfide intermediate during folding. Much of the remaining one-third of the polypeptide chain, in particular the N-terminal 14 residues, is largely disordered; this accounts for the ability of this intermediate to form readily any of the three possible second disulfide bonds involving Cys-5, -14, and -38. The partially folded conformation of this intermediate provides direct evidence for the importance of native-like interactions between elements of secondary structure in directing protein folding, which is assumed in many studies.

A two-dimensional 1H NMR study on Megasphaera elsdenii flavodoxin in the reduced state. Sequential assignments.

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 degrees C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against 2H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the 1H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contracts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin.

Solution conformation of a peptide fragment representing a proposed RNA-binding site of a viral coat protein studied by two-dimensional NMR.

The first 25 amino acids of the coat protein of cowpea chlorotic mottle virus are essential for binding the encapsidated RNA. Although an alpha-helical conformation has been predicted for this highly positively charged N-terminal region [Argos, P. (1981) Virology 110, 55-62; Vriend, G., Verduin, B. J. M., & Hemminga, M. A. (1986) J. Mol. Biol. 191, 453-460], no experimental evidence for this conformation has been presented so far. In this study, two-dimensional proton NMR experiments were performed on a chemically synthesized pentacosapeptide containing the first 25 amino acids of this coat protein [Ten Kortenaar, P. B. W., Krüse, J., Hemminga, M. A., & Tesser, G. I. (1986) Int. J. Pept. Protein Res. 27, 401-413]. All resonances could be assigned by a combined use of two-dimensional correlated spectroscopy and nuclear Overhauser enhancement spectroscopy carried out at four different temperatures. Various NMR parameters indicate the presence of a conformational ensemble consisting of helical structures rapidly converting into more extended states. Differences in chemical shifts and nuclear Overhauser effects indicate that lowering the temperature induces a shift of the dynamic equilibrium toward more helical structures. At 10 degrees C, a perceptible fraction of the conformational ensemble consists of structures with an alpha-helical conformation between residues 9 and 17, likely starting with a turnlike structure around Thr9 and Arg10. Both the conformation and the position of this helical region agree well with the secondary structure predictions mentioned above.