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PubMed2006

Two crystal structures of Trichoderma reesei hydrophobin HFBI--the structure of a protein amphiphile with and without detergent interaction.

Hydrophobins are small fungal proteins that are highly surface active and possess a unique ability to form amphiphilic membranes through spontaneous self-assembly. The first crystal structure of a hydrophobin, Trichoderma reesei HFBII, revealed the structural basis for the function of this amphiphilic protein--a patch consisting of hydrophobic side chains on the protein surface. Here, the crystal structures of a native and a variant T. reesei hydrophobin HFBI are presented, revealing the same overall structure and functional hydrophobic patch as in the HFBII structure. However, some structural flexibility was found in the native HFBI structure: The asymmetric unit contained four molecules, and, in two of these, an area of seven residues was displaced as compared to the two other HFBI molecules and the previously determined HFBII structure. This structural change is most probably induced by multimer formation. Both the native and the N-Cys-variant of HFBI were crystallized in the presence of detergents, but an association between the protein and a detergent was only detected in the variant structure. There, the molecules were arranged into an extraordinary detergent-associated octamer and the solvent content of the crystals was 75%. This study highlights the conservation of the fold of class II hydrophobins in spite of the low sequence identity and supports our previous suggestion that concealment of the hydrophobic surface areas of the protein is the driving force in the formation of multimers and monolayers in the self-assembly process.

Amino Acid Sequence↗

PubMed2002

Molecules for materials: structures, thermochemistry, and electron affinities of the digermanium fluorides Ge2Fn/Ge2Fn- (n = 1-6): a wealth of unusual structures.

A systematic investigation of Ge2Fn/Ge2Fn- systems was carried out with five density functional theory (DFT) methods in conjunction with DZP++ basis sets. For each compound various structures, including minima, transition states and other energetically low lying stationary points, were optimized. The geometries and relative energies are discussed and compared. Adiabatic electron affinities, vertical electron affinities and anion vertical detachment energies are reported. Three types of dissociation energies pertaining to the global minima for each compound are reported. The theoretical predictions are in good agreement with the limited available experimental results. Many unusual structural features are predicted for these systems. Neutral Ge2F is predicted to have a bridged C2v structure, while its anion is very floppy, with the bridged structure very slightly favoured. The Ge2F2 molecule is predicted to have the butterfly structure known from experiment for Si2H2, while the Ge2F3- ion has a trans-bent structure. Ge2F3 is predicted to have an unprecedented FGe-F-GeF structure with no Ge-Ge bond, while its anion has a somewhat more conventional monobridged structure, analogous to that of the nonclassical vinyl cation. Neutral Ge2F4 has a dibridged structure of C2h symmetry, while its anion has a trans-bent structure with a very long Ge-Ge bond. The Ge2F5 molecule is doubly bridged and has no Ge-Ge bond, while the anion is of the type F2Ge-F-GeF2, again with no Ge-Ge bond. Ge2F6 has the anticipated ethane structure, as does its anion, but with a very long Ge-Ge bond. The adiabatic electron affinities (EAad) are predicted to be 2.12 (Ge2F), 2.03 (Ge2F2), 2.02 (Ge2F3), 1.64 (Ge2F4), 4.57 (Ge2F5), and 2.66 eV (Ge2F6), respectively, by the BHLYP method, which is regarded as the best method in the present paper for predicting EAs. Comparisons with the analogous C2Fn and Si2Fn systems reveals some interesting trends and differences. For example, while C2F6 will not capture an electron, Si2F6 is predicted to have small electron affinity (0.73 eV), while that of Ge2F6 is substantial. The same trend to larger EAs on going down the Periodic Table is seen for the X2F5 systems, with 1.77 (C2F5), 2.68 (Si2F5), and 4.57 eV (Ge2F5). However, the EAs do not follow a monotonic trend for the X2F, X2F3 and X2F4 systems with respect to the series X = C, Si, Ge.

Journal Article↗

PubMed1995

Significance of structural changes in proteins: expected errors in refined protein structures.

A quantitative expression key to evaluating significant structural differences or induced shifts between any two protein structures is derived. Because crystallography leads to reports of a single (or sometimes dual) position for each atom, the significance of any structural change based on comparison of two structures depends critically on knowing the expected precision of each median atomic position reported, and on extracting it for each atom, from the information provided in the Protein Data Bank and in the publication. The differences between structures of protein molecules that should be identical, and that are normally distributed, indicating that they are not affected by crystal contacts, were analyzed with respect to many potential indicators of structure precision, so as to extract, essentially by "machine learning" principles, a generally applicable expression involving the highest correlates. Eighteen refined crystal structures from the Protein Data Bank, in which there are multiple molecules in the crystallographic asymmetric unit, were selected and compared. The thermal B factor, the connectivity of the atom, and the ratio of the number of reflections to the number of atoms used in refinement correlate best with the magnitude of the positional differences between regions of the structures that otherwise would be expected to be the same. These results are embodied in a six-parameter equation that can be applied to any crystallographically refined structure to estimate the expected uncertainty in position of each atom. Structure change in a macromolecule can thus be referenced to the expected uncertainty in atomic position as reflected in the variance between otherwise identical structures with the observed values of correlated parameters.

Crystallization↗

PubMed2002

Human brain potentials reveal similar processing of non-linguistic abstract structure and linguistic syntactic structure.

This research tested the hypothesis that shared or related neurophysiological processes are required for treating (a) non-linguistic abstract structure and (b) some aspects of linguistic syntactic structure. In language, one syntactic structure can be used to create an open class of sentences. We have previously proposed a relation between this generative aspect of syntactic structure and the abstract structure of non-linguistic sequences. For instance, the sequences 'ABCBAC' and 'DEFEDF' have different serial order or serial structure, but share the same abstract structure '123213'. Our recent studies of neuropsychology, simulation and ERPs argued that similar neurophysiological processes are involved in treating non-linguistic abstract structure and certain aspects of linguistic syntactic structure. The current research tests this hypothesis by examining the ERP profile evoked during the processing of non-linguistic sequences vs. sentences. Ten healthy subjects were trained to discriminate between syntactically correct and incorrect sequences and sentences presented visually on a video screen. During the subsequent ERP recording, subjects discriminated between correct and incorrect sequences and sentences presented visually on the screen. This discrimination task yielded, for anomalies in both the abstract and syntactic conditions, a late positivity around 550 ms with partially overlapping topography. These results support our hypothesis that shared or related neurophysiological processes are required for treating non-linguistic abstract structure and aspects of linguistic syntactic structure. However, they also suggest that the overlap between these two types of processing is not complete.

Adult↗

PubMed1998

How representative are the known structures of the proteins in a complete genome? A comprehensive structural census.

BACKGROUND: Determining how representative the known structures are of the proteins encoded by a complete genome is important for assessing to what extent our current picture of protein stability and folding is overly influenced by biases in the structure databank (PDB). It is also important for improving database-based methods of structure prediction and genome annotation. RESULTS: The known structures are compared to the proteins encoded by eight complete microbial genomes in terms of simple statistics such as sequence length, composition and secondary structure. The known structures are represented by a collection of nonhomologous domains from the PDB and a smaller list of 'biophysical proteins' on which folding experiments have concentrated. The proteins encoded by the genomes are considered as a whole and divided into various regions, such as known-structure homologue, low complexity (nonglobular), transmembrane or linker. Various tests are performed to assess the significance of the reported differences, in both a practical and a statistical sense. CONCLUSIONS: The proteins encoded by the genomes are significantly different from those in the PDB. Their sequence lengths, which follow an extreme value distribution, are longer than the PDB proteins and much longer than the biophysical proteins. Their composition differs from the PDB proteins in having more Lys, Ile, Asn and Gln and less Cys and Trp. This is true overall and especially for the regions corresponding to soluble proteins of as yet unknown fold. Secondary-structure prediction on these uncharacterized regions indicates that they contain on average more helical structure than the PDB; differences about this mean are small, with yeast having slightly more sheet structure and Haemophilus influenzae and Helicobacter pylori more helical structure. Further information is available through the GeneCensus system at http://bioinfo.mbb.yale.edu/genome.

Animals↗

PubMed2003

On the molecular basis of the recognition of angiotensin II (AII). NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131.

The high-resolution 3D structure of the octapeptide hormone angiotensin II (AII) in aqueous solution has been obtained by simulated annealing calculations, using high-resolution NMR-derived restraints. After final refinement in explicit water, a family of 13 structures was obtained with a backbone RMSD of 0.73 +/- 0.23 A. AII adopts a fairly compact folded structure, with its C-terminus and N-terminus approaching to within approximately 7.2 A of each other. The side chains of Arg2, Tyr4, Ile5 and His6 are oriented on one side of a plane defined by the peptide backbone, and the Val3 and Pro7 are pointing in opposite directions. The stabilization of the folded conformation can be explained by the stacking of the Val3 side chain with the Pro7 ring and by a hydrophobic cluster formed by the Tyr4, Ile5 and His6 side chains. Comparison between the NMR-derived structure of AII in aqueous solution and the refined crystal structure of the complex of AII with a high-affinity mAb (Fab131) [Garcia, K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T., Amzel, L.M. (1992) Science257, 502-507] provides important quantitative information on two common structural features: (a) a U-shaped structure of the Tyr4-Ile5-His6-Pro7 sequence, which is the most immunogenic epitope of the peptide, with the Asp1 side chain oriented towards the interior of the turn approaching the C-terminus; (b) an Asx-turn-like motif with the side chain aspartate carboxyl group hydrogen-bonded to the main chain NH group of Arg2. It can be concluded that small rearrangements of the epitope 4-7 in the solution structure of AII are required by a mean value of 0.76 +/- 0.03 A for structure alignment and approximately 1.27 +/- 0.02 A for sequence alignment with the X-ray structure of AII bound to the mAb Fab131. These data are interpreted in terms of a biological "nucleus" conformation of the hormone in solution, which requires a limited number of structural rearrangements for receptor-antigen recognition and binding.

Angiotensin II↗

PubMed1999

A comparison of sequence and structure protein domain families as a basis for structural genomics.

MOTIVATION: Protein families can be defined based on structure or sequence similarity. We wanted to compare two protein family databases, one based on structural and one on sequence similarity, to investigate to what extent they overlap, the similarity in definition of corresponding families, and to create a list of large protein families with unknown structure as a resource for structural genomics. We also wanted to increase the sensitivity of fold assignment by exploiting protein family HMMs. RESULTS: We compared Pfam, a protein family database based on sequence similarity, to Scop, which is based on structural similarity. We found that 70% of the Scop families exist in Pfam while 57% of the Pfam families exist in Scop. Most families that occur in both databases correspond well to each other, but in some cases they are different. Such cases highlight situations in which structure and sequence approaches differ significantly. The comparison enabled us to compile a list of the largest families that do not occur in Scop; these are suitable targets for structure prediction and determination, and may be useful to guide projects in structural genomics. It can be noted that 13 out of the 20 largest protein families without a known structure are likely transmembrane proteins. We also exploited Pfam to increase the sensitivity of detecting homologs of proteins with known structure, by comparing query sequences to Pfam HMMs that correspond to Scop families. For SWISSPROT+TREMBL, this yielded an increase in fold assignment from 31% to 42% compared to using FASTA only. This method assigned a structure to 22% of the proteins in Saccharomyces cerevisiae, 24% in Escherichia coli, and 16% in Methanococcus jannaschii.

Computational Biology↗

PubMed1996

Crystal structure of glutathione synthetase at optimal pH: domain architecture and structural similarity with other proteins.

The crystal structure of Escherichia coli B glutathione synthetase (GSHase) has been determined at the optimal catalytic condition pH 7.5. The most significant structural difference from the structure at pH 6.0 is the movement of the central domain towards the N-terminal domain almost as a rigid body. As a result of this movement, new interdomain and intersubunit polar interactions are formed which stabilize the dimeric structure further. The structure of GSHase at optimal pH was compared with 294 other known protein structures in terms of the spatial arrangements of secondary structural elements. Three enzymes (D-alanine: D-alanine ligase, succinyl-CoA synthetase and the biotin carboxylase subunit of acetyl-CoA carboxylase) were found to have structures similar to the ATP-binding site of GSHase, which extends across two domains. The ATP-binding sites in these four enzymes are composed of two antiparallel beta-sheets and are different from the classic mononucleotide-binding fold. Except for these proteins, no significant structural similarity was detected between GSHase and the other ATP-binding proteins. A structural motif in the N-terminal domain of GSHase has been found to be similar to the NAD-binding fold. This structural motif is shared by a number of other proteins that bind various negatively charged molecules.

Acetyl-CoA Carboxylase↗

PubMed2005

Comparing hypothetical structures generated in the third Cambridge blind test of crystal structure prediction.

In the third Cambridge blind test of crystal structure prediction, participants submitted extended lists of up to 100 hypothetical structures. In this paper these lists are analyzed for the two small semi-rigid molecules, hydantoin and azetidine, by performing a new energy minimization using an accurate force field, and grouping these newly minimized structures into clusters of equivalent structures. Many participants found the same low-energy structures, but no list appeared to be complete even for the structures with one independent molecule in the asymmetric unit. This may well be due to the fact that a cutoff at even 100 structures cannot ensure the presence of a structure that has a relatively high ranking in another force field. Moreover, some structures should have possibly been discarded because they correspond to transition states rather than true energy minima. The r.m.s. deviation between energies in corresponding clusters was calculated to compare the reported relative crystal energies for each pair of participants. Some groups of force fields show a reasonably good correspondence, yet the order of magnitude of their discrepancies is comparable to the energy differences between, say, the first ten structures of lowest energy. Therefore, even if we assume that energy is a sufficient criterion, it is not surprising that crystal structure predictions are still inconsistent and unreliable.

Algorithms↗

PubMed2002

Protein structure prediction constrained by solution X-ray scattering data and structural homology identification.

Here we perform a systematic exploration of the use of distance constraints derived from small angle X-ray scattering (SAXS) measurements to filter candidate protein structures for the purpose of protein structure prediction. This is an intrinsically more complex task than that of applying distance constraints derived from NMR data where the identity of the pair of amino acid residues subject to a given distance constraint is known. SAXS, on the other hand, yields a histogram of pair distances (pair distribution function), but the identities of the pairs contributing to a given bin of the histogram are not known. Our study is based on an extension of the Levitt-Hinds coarse grained approach to ab initio protein structure prediction to generate a candidate set of C(alpha) backbones. In spite of the lack of specific residue information inherent in the SAXS data, our study shows that the implementation of a SAXS filter is capable of effectively purifying the set of native structure candidates and thus provides a substantial improvement in the reliability of protein structure prediction. We test the quality of our predicted C(alpha) backbones by doing structural homology searches against the Dali domain library, and find that the results are very encouraging. In spite of the lack of local structural details and limited modeling accuracy at the C(alpha) backbone level, we find that useful information about fold classification can be extracted from this procedure. This approach thus provides a way to use a SAXS data based structure prediction algorithm to generate potential structural homologies in cases where lack of sequence homology prevents identification of candidate folds for a given protein. Thus our approach has the potential to help in determination of the biological function of a protein based on structural homology instead of sequence homology.

Computational Biology↗

PubMed2006

Comparison of homology models and X-ray structures of the nuclear receptor CAR: assessing the structural basis of constitutive activity.

The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural basis has been analysed during the last decade. Recently, we published a homology model of the CAR ligand binding domain (LBD) based on the X-ray structures of the closely related pregnane X (PXR) and vitamin D (VDR) receptor. A detailed analysis of the homology model and molecular dynamics (MD) simulations afforded us to propose a potential mechanism underlying the constitutive activity of CAR. Almost simultaneously, X-ray structures of human and mouse CAR LBD were released. In the present study, a detailed analysis and comparison of homology model and X-ray structures is carried out in order to evaluate the quality and reliability of our homology modelling procedure. The hypothesis of the constitutive activity which we proposed on the basis of our modelling results was tested for consistency with the crystal structures. In addition, the features stated to be essential for the basal activity based on the X-ray data were investigated by means of molecular dynamics simulations. Our results show that the homology modelling procedure was able to predict the CAR LBD structure with high accuracy. Structural features that have been revealed as critical for constitutive activity in the model are also observed in the X-ray structures. Furthermore, the MD simulations of the CAR X-ray structures and a detailed analysis of other NRs clarify the role of distinct structural features that have been assigned an important role for the constitutive activity.

Amino Acid Sequence↗

PubMed2005

Redox-dependent structural differences in putidaredoxin derived from homologous structure refinement via residual dipolar couplings.

Structural differences in the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), from the camphor hydroxylation pathway of Pseudomonas putida have been investigated as a function of oxidation state of the iron cluster. Pdx is involved in biological electron transfer to cytochrome P450(cam) (CYP101). Redox-dependent differences have been observed previously for Pdx in terms of binding affinities to CYP101, NMR spectral differences, and dynamic properties. To further characterize these differences, structure refinement of both oxidized and reduced Pdx has been carried out using a hybrid approach utilizing paramagnetic distance restraints and NMR orientational restraints in the form of backbone (15)N residual dipolar couplings. Use of these new restraints has improved the structure of oxidized Pdx considerably over the earlier solution NMR structure without RDC restraints, with the new structure now much closer in overall fold to the recently published X-ray crystal structures. We now observe better defined relative orientations of the major secondary structure elements as also of the conformation of the metal binding loop region. Extension of this approach to structure calculation of reduced Pdx has identified structural differences that are primarily localized for residues in the C-terminal interaction domain consisting of the functionally important residue Trp 106 and regions near the metal binding loop in Pdx. These redox-dependent structural differences in Pdx correlate to dynamic changes observed before and may be linked to differences in binding and electron transfer properties between oxidized and reduced Pdx.

Crystallography, X-Ray↗

PubMed2006

Flexible Structural Neighborhood--a database of protein structural similarities and alignments.

Protein structures are flexible, changing their shapes not only upon substrate binding, but also during evolution as a collective effect of mutations, deletions and insertions. A new generation of protein structure comparison algorithms allows for such flexibility; they go beyond identifying the largest common part between two proteins and find hinge regions and patterns of flexibility in protein families. Here we present a Flexible Structural Neighborhood (FSN), a database of structural neighbors of proteins deposited in PDB as seen by a flexible protein structure alignment program FATCAT, developed previously in our group. The database, searchable by a protein PDB code, provides lists of proteins with statistically significant structural similarity and on lower menu levels provides detailed alignments, interactive superposition of structures and positions of hinges that were identified in the comparison. While superficially similar to other structural protein alignment resources, FSN provides a unique resource to study not only protein structural similarity, but also how protein structures change. FSN is available from a server http://fatcat.burnham.org/fatcat/struct_neighbor and by direct links from the PDB database.

Databases, Protein↗

PubMed1997

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.

Amino Acid Sequence↗

PubMed2000

Structural basis for the network of functional cooperativities in cytochrome c(3) from Desulfovibrio gigas: solution structures of the oxidised and reduced states.

Cytochrome c(3) is a 14 kDa tetrahaem protein that plays a central role in the bioenergetic metabolism of Desulfovibrio spp. This involves an energy transduction mechanism made possible by a complex network of functional cooperativities between redox and redox/protolytic centres (the redox-Bohr effect), which enables cytochrome c(3) to work as a proton activator. The three-dimensional structures of the oxidised and reduced Desulfovibrio gigas cytochrome c(3) in solution were solved using 2D (1)H-NMR data. The reduced protein structures were calculated using INDYANA, an extended version of DYANA that allows automatic calibration of NOE data. The oxidised protein structure, which includes four paramagnetic centres, was solved using the program PARADYANA, which also includes the structural paramagnetic parameters. In this case, initial structures were used to correct the upper and lower volume restraints for paramagnetic leakage, and angle restraints derived from (13)C Fermi contact shifts of haem moiety substituents were used for the axial histidine ligands. Despite the reduction of the NOE intensities by paramagnetic relaxation, the final family of structures is of similar precision and accuracy to that obtained for the reduced form. Comparison of the two structures shows that, although the global folds of the two families of structures are similar, significant localised differences occur upon change of redox state, some of which could not be detected by comparison with the X-ray structure of the oxidised state: (1) there is a redox-linked concerted rearrangement of Lys80 and Lys90 that results in the stabilisation of haem moieties II and III when both molecules are oxidised or both are reduced, in agreement with the previously measured positive redox cooperativity between these two haem moieties. This cooperativity regulates electron transfer, enabling a two-electron step adapted to the function of cytochromes c(3) as the coupling partner of hydrogenase; and (2) the movement of haem I propionate 13 towards the interior of the protein upon reduction explains the positive redox-Bohr effect, establishing the structural basis for the redox-linked proton activation mechanism necessary for energy conservation, driving ATP synthesis.

Allosteric Regulation↗

PubMed2005

Progress of structural genomics initiatives: an analysis of solved target structures.

The explosion in gene sequence data and technological breakthroughs in protein structure determination inspired the launch of structural genomics (SG) initiatives. An often stated goal of structural genomics is the high-throughput structural characterisation of all protein sequence families, with the long-term hope of significantly impacting on the life sciences, biotechnology and drug discovery. Here, we present a comprehensive analysis of solved SG targets to assess progress of these initiatives. Eleven consortia have contributed 316 non-redundant entries and 323 protein chains to the Protein Data Bank (PDB), and 459 and 393 domains to the CATH and SCOP structure classifications, respectively. The quality and size of these proteins are comparable to those solved in traditional structural biology and, despite huge scope for duplicated efforts, only 14% of targets have a close homologue (>/=30% sequence identity) solved by another consortium. Analysis of CATH and SCOP revealed the significant contribution that structural genomics is making to the coverage of superfamilies and folds. A total of 67% of SG domains in CATH are unique, lacking an already characterised close homologue in the PDB, whereas only 21% of non-SG domains are unique. For 29% of domains, structure determination revealed a remote evolutionary relationship not apparent from sequence, and 19% and 11% contributed new superfamilies and folds. The secondary structure class, fold and superfamily distributions of this dataset reflect those of the genomes. The domains fall into 172 different folds and 259 superfamilies in CATH but the distribution is highly skewed. The most populous of these are those that recur most frequently in the genomes. Whilst 11% of superfamilies are bacteria-specific, most are common to all three superkingdoms of life and together the 316 PDB entries have provided new and reliable homology models for 9287 non-redundant gene sequences in 206 completely sequenced genomes. From the perspective of this analysis, it appears that structural genomics is on track to be a success, and it is hoped that this work will inform future directions of the field.

Animals↗

PubMed1996

The solution structure of the first zinc finger domain of SWI5: a novel structural extension to a common fold.

BACKGROUND: The 2Cys-2His (C2-H2) zinc finger is a protein domain commonly used for sequence-specific DNA recognition. The zinc fingers of the yeast transcription factors SWI5 and ACE2 share strong sequence homology, which extends into a region N-terminal to the first finger, suggesting that the DNA-binding domains of these two proteins include additional structural elements. RESULTS: Structural analysis of the zinc fingers of SWI5 reveals that a 15 residue region N-terminal to the finger motifs forms part of the structure of the first finger domain, adding a beta strand and a helix not previously observed in other zinc finger structures. Sequence analysis suggests that other zinc finger proteins may also have this structure. Biochemical studies show that this additional structure increases DNA-binding affinity. CONCLUSIONS: The structural analysis presented reveals a novel zinc finger structure in which additional structural elements have been added to the C2-H2 zinc finger fold. This additional structure may enhance stability and has implications for DNA recognition by extending the potential DNA-binding surface of a single zinc finger domain.

Amino Acid Sequence↗

PubMed1996

Structure and dynamics of a designed helix-loop-helix dimer in dilute aqueous trifluoroethanol solution. A strategy for NMR spectroscopic structure determination of molten globules in the rational design of native-like proteins.

BACKGROUND: The overwhelming majority of engineered amino acid sequences designed to fold into well defined tertiary structures show the hallmarks of molten globules. Although imperfectly folded, the structures of these polypeptides are of considerable interest in assessing the predictive power of design strategies and in understanding the structural basis for the formation of proteins with native-like properties. This paper describes a strategy for the structural characterization of molten globules by NMR spectroscopy applied to the study of SA-42, a polypeptide with 42 amino acids that folds into a hairpin helix-loop-helix dimer. RESULTS: The 1H NMR spectrum of SA-42 was assigned in several mixtures of water and trifluoroethanol (TFE) (0-30 vol%) and small amounts of TFE were shown to have a significant effect on the spectrum. The secondary and supersecondary structures of SA-42 were determined. In aqueous solution a helix-loop-helix dimer is formed, but in 30 vol% of TFE the population of hairpin dimers are negligible and SA-42 is monomeric, folding into two non-interacting helical segments. In solutions containing less than 3 vol% of TFE the structure is very similar to that in water and the structural information may be used to develop the motif in aqueous solution. Less well ordered amino acid residue sidechains in the hydrophobic core were identified. Helix distortion in the tetrahelix bundle was found to be small. CONCLUSIONS: Detailed information about molten globule structures in aqueous solution can be obtained from NMR spectroscopy if the spectra are assigned in dilute TFE solution. On the basis of the NMR spectroscopic analysis, the solution structure of SA-42 was found to be close to the designed one. A route for developing native-like properties in SA-42 is suggested based on the identification by NMR spectroscopy of some less well ordered amino acid sidechains in the hydrophobic core and on the observed structural rigidity of the two helices.

Amino Acid Sequence↗