An approach to global fold determination using limited NMR data from larger proteins selectively protonated at specific residue types

Summary A combination of calculation and experiment is used to demonstrate that the global fold of larger proteins can be rapidly determined using limited NMR data. The approach involves a combination of heteronuclear triple resonance NMR experiments with protonation of selected residue types in an otherwise completely deuterated protein. This method of labelling produces proteins with -specific deuteration in the protonated residues, and the results suggest that this will improve the sensitivity of experiments involving correlation of side-chain (¹H and ¹³C) and backbone (¹H and ¹⁵N) amide resonances. It will allow the rapid assignment of backbone resonances with high sensitivity and the determination of a reasonable structural model of a protein based on limited NOE restraints, an application that is of increasing importance as data from the large number of genome sequencing projects accumulates. The method that we propose should also be of utility in extending the use of NMR spectroscopy to determine the structures of larger proteins.The first two authors contributed equally to this work.     

Solution structure of chicken liver basic fatty acid binding protein

Chicken liver basic fatty acid binding protein (Lb-FABP) belongs to the basic-type fatty acid binding proteins, a novel group of proteins isolated from liver of different non mammalian species whose structure is not known. The structure of Lb-FABP has been solved by ¹H NMR. The overall fold of Lb-FABP, common to the other proteins of the family, consists of ten antiparallel -strands organised in two nearly ortogonal -sheets with two alpha helices closing the protein cavity where small hydrophobic ligands can be bound. The binding specificity of the protein is not known, however, based on the high sequence and structural similarity with an orthologous protein, ileal lipid binding protein, it is suggested that bile acids may be the putative ligands.     

Fully automated high-quality NMR structure determination of small <Superscript>2</Superscript>H-enriched proteins

Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 Å, an all atom RMSD value of 1.2 Å to the crystal structure for well-defined regions, and RMSD value of 1.1 Å to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with ¹⁵N- and ¹³C-edited NOESY data obtained with a perdeuterated ¹⁵N, ¹³C-enriched ¹³CH₃ methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR.     

Complete protein structure determination using backbone residual dipolar couplings and sidechain rotamer prediction

Residual dipolar couplings provide significant structural information for proteins in the solution state, which makes them attractive for the rapid determination of protein structures. While dipolar couplings contain inherent structural ambiguities, these can be reduced via an overlap similarity measure that insists that protein fragments assigned to overlapping regions of the sequence must have self-consistent structures. This allows us to determine a backbone fold (including the correct C–C bond orientations) using only residual dipolar coupling data from one ordering medium. The resulting backbone structures are of sufficient quality to allow for modeling of sidechain rotamer states using a rotamer prediction algorithm and a force field employing the Surface Generalized Born continuum solvation model. We demonstrate the applicability of the method using experimental data for ubiquitin. These results illustrate the synergies that are possible between protein structural database and molecular modeling methods and NMR spectroscopy, and we expect that the further development of these methods will lead to the extraction of high resolution structural information from minimal NMR data.     

Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints

We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N_i-H^N _i, N_i-C_i–1, H^N _i - C_i–1) in two tensor frames and only backbone H^N-H^N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 Å with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms.     

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when ¹³C^β and ¹³C′ shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using ¹³C^α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6

Summary Essentially complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignments for the 185-aminoacid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [¹⁵N]-and [¹⁵N, ¹³C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of ¹³C-chemical shift, amide hydrogen-bond exchange, and ¹⁵N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five -helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.     

Protein φ and ψ dihedral restraints determined from multidimensional hypersurface correlations of backbone chemical shifts and their use in the determination of protein tertiary structures

The chemical shifts of the backbone atoms of proteins can be used to obtainrestraints that can be incorporated into structure determination methods. Eachchemical shift can be used to define a restraint and these restraints can besimultaneously used to define the local, secondary structure features. Theglobal fold can be determined by a combined use of the chemical shift basedrestraints along with the long-range information present in the NOEs ofpartially deuterated proteins or the amide–amide NOEs but not from suchlimited NOE data sets alone. This approach has been demonstrated to be capableof determining the overall folding pattern of four proteins. This suggeststhat solution-state NMR methods can be extended to the structure determinationof larger proteins by using the information present in the chemical shifts ofthe backbone atoms along with the data that can be obtained on a small numberof labeled forms.     

Competitive cobalt for zinc substitution in mammalian methionine sulfoxide reductase B1 overexpressed in E. coli: structural and functional insight

Expression of the mammalian enzyme methionine sulfoxide reductase B1 (MsrB1) in Escherichia coli growing in cobalt-containing media resulted in the reproducible appearance of the stable cobalt-containing protein MsrB1-Co. NMR studies and biocomputing using the programs AnisoFit and Amber allowed us to generate a structure of MsrB1-Co sharing the overall fold with the native zinc-containing protein MsrB1-Zn. Our data suggest that the N-terminus containing resolving cysteine tends to be closer to the protein’s catalytic center than was previously reported. It is argued that this proximity supports the proposed catalytic mechanism and ensures high catalytic efficiency of MsrB1. Functional studies showed that both MsrB1-Zn and MsrB1-Co exhibit similar levels of activity, in agreement with the structural studies performed. The proposed metal ion substitution approach may have a methodological significance in determining whether methionine sulfoxide reductase B proteins contain a metal ion.     

Optimized set of two-dimensional experiments for fast sequential assignment,secondary structure determination,and backbone fold validation of 13C/15N-labelled proteins

NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional ¹H-¹⁵N HSQC-like spectra. The ¹H-¹⁵N correlation peaks are frequency shifted by an amount ± _X along the ¹⁵N dimension, where _X is the C, C, or H frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.     

A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: Application to mastoparan X solubilized in n-octylglucoside

Summary Application of ¹H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a ¹H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by ¹H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the ¹H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein ¹H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and -protons. Regioselective COSY spectra provide scalar coupling constants between amide and -protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants. It is shown that mastoparan adopts an -helical conformation when bound to nonionic detergent micelles. The present method is expected to increase the applicability of ¹H solution NMR methods to membrane proteins and peptides.Abbreviations 2D NMR two-dimensional NMR - COSY correlated spectroscopy - DANTE delays alternating nutations for tailored excitation - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy     

Structural and Functional Similarity between the Bacterial Type III Secretion System Needle Protein PrgI and the Eukaryotic Apoptosis Bcl-2 Proteins

Background

Functional similarity is challenging to identify when global sequence and structure similarity is low. Active-sites or functionally relevant regions are evolutionarily more stable relative to the remainder of a protein structure and provide an alternative means to identify potential functional similarity between proteins. We recently developed the FAST-NMR methodology to discover biochemical functions or functional hypotheses of proteins of unknown function by experimentally identifying ligand binding sites. FAST-NMR utilizes our CPASS software and database to assign a function based on a similarity in the structure and sequence of ligand binding sites between proteins of known and unknown function.

Methodology/Principal Findings

The PrgI protein from Salmonella typhimurium forms the needle complex in the type III secretion system (T3SS). A FAST-NMR screen identified a similarity between the ligand binding sites of PrgI and the Bcl-2 apoptosis protein Bcl-xL. These ligand binding sites correlate with known protein-protein binding interfaces required for oligomerization. Both proteins form membrane pores through this oligomerization to release effector proteins to stimulate cell death. Structural analysis indicates an overlap between the PrgI structure and the pore forming motif of Bcl-xL. A sequence alignment indicates conservation between the PrgI and Bcl-xL ligand binding sites and pore formation regions. This active-site similarity was then used to verify that chelerythrine, a known Bcl-xL inhibitor, also binds PrgI.

Conclusions/Significance

A structural and functional relationship between the bacterial T3SS and eukaryotic apoptosis was identified using our FAST-NMR ligand affinity screen in combination with a bioinformatic analysis based on our CPASS program. A similarity between PrgI and Bcl-xL is not readily apparent using traditional global sequence and structure analysis, but was only identified because of conservation in ligand binding sites. These results demonstrate the unique opportunity that ligand-binding sites provide for the identification of functional relationships when global sequence and structural information is limited.     

Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.High-resolution structures of proteins are essential for understanding cellular processes. Determining protein structures, however, is difficult: protein stability, purity, quantity, and solubility critically affect success. Nuclear magnetic resonance (NMR)¹ spectroscopy can only be applied to proteins of limited size, whereas x-ray crystallography necessitates prior crystallization of the protein. These conditions make structure determination challenging for many proteins of biological relevance. This includes especially proteins that contain intrinsically unstructured or long coiled-coil regions, proteins associated to a membrane (1, 2) or parts of multi-protein complexes (3). New developments to overcome some of these restrictions include x-ray free electron lasers (XFEL) (4), which only require microcrystals, new detectors in cryo-electron microscopy (5) and in-cell NMR (6), which analyzes the structure of small proteins in a cellular context. Further advancements that assist with protein structure determination have included the development of being able to use sparse NMR data, for example using backbone only data (7), and the understanding of evolutionary constraints for predicting protein structure (8).We present a novel approach to obtain structural details of proteins by mass spectrometry. This can be accomplished through cross-linking and mass spectrometry (CLMS) (9–11). Cross-links establish covalent bonds between residue pairs close in space but not necessarily in sequence. This conserves structural information throughout an analysis that follows the standard proteomics workflow. Typically, a bi-functional chemical reagent, the cross-linker, is incubated with a protein of interest. The cross-linker reacts with two residues—often involving the side-chain amine of lysine—that are near each other in the folded protein. A protease such as trypsin is used to degrade the protein. The resulting mix of cross-linked peptides is then analyzed by mass spectrometry and database searching akin to other shotgun proteomics approaches (12). The pairs of cross-linked residues are identified from the mass spectrometric data and provide information on which residues are near each other in the folded protein. This information is represented in the form of distance constraints, deducible from the length of the cross-linking agent.CLMS data has been used to study large multi-protein complexes (13), networks (14) and proteins in whole cells (15). The distance constraints obtained are sparse but complement other structural data in integrated structural biology well (10). Cross-link data allow placing high-resolution structures of individual sub-units in the electron microscopy structure of an assembled multi-protein complex to obtain its quasi-atomic resolution structure, e.g. the proteasome (16). In an alternative approach, genetic site-directed positioning of a photo-reactive group, azide, as part of a phenylalanine analog, was recently used to derive proximity information that allowed modeling of receptor CRF1R bound to its native ligand (17). Young et al. used 15 cross-links to identify the correct fold of bovine basic fibroblast growth factor using threading and homology modeling (18). In a similar study, Singh et al. used eight cross-links to build a monomer homology model of the major capsid protein E of bacteriophage lambda and to derive a pseudoatomic model of the lambda procapsid shell (19). In both of the aforementioned cases, the cross-link information was merely used to verify structural models by threading and homology modeling, and did not significantly impact model building. Prior attempts to leverage cross-linking data in structure determination delivered improvements, however, without leading to high-resolution models (20).Here, we increase the spatial resolution of information obtained through cross-linking by using a highly reactive chemical as a cross-linking agent. This broadens the specificity of cross-linking and thus increases the spatial resolution in conjunction with mass spectrometry. We employ the heterobifunctional chemical cross-linker sulfosuccinimidyl 4,4′-azipentanoate, sulfo-SDA (21), to chemically cross-link a protein, human serum albumin (HSA).We combine the distance constraints provided by cross-linking and mass spectrometry with computational, conformational space search. This approach allows us to generate structural models of HSA domains that correlate highly with the structure of HSA solved by x-ray crystallography. With this method, we show that our pipeline can be used to analyze the structure of HSA domains from HSA not only in it''s purified form, but additionally unpurified and in its native environment, human blood serum.     

Rapid,robotic, small‐scale protein production for NMR screening and structure determination

Three‐dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time‐consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi‐automated protocol for isotopically‐labeled protein production using the Maxwell‐16, a commercially available bench top robot, that allows for single‐step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different ¹⁵N‐labeled proteins, accelerating the validation process by more than 10‐fold. The yield from a single channel of the Maxwell‐16 is sufficient for acquisition of a high‐quality 2D ¹H‐¹⁵N‐HSQC spectrum using a 3‐mm sample cell and 5‐mm cryogenic NMR probe. Maxwell‐16 screening of a control group of proteins reproduced previous validation results from conventional small‐scale expression screening and large‐scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par‐3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U‐¹⁵N,¹³C] protein prepared using the Maxwell‐16. This novel semi‐automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale‐up steps.     

Refinement of the solution structure of the heparin-binding domain of vascular endothelial growth factor using residual dipolar couplings

Previous NMR structural studies of the heparin-binding domain of vascular endothelial growth factor (VEGF₁₆₅) revealed a novel fold comprising two subdomains, each containing two disulfide bridges and a short two-stranded antiparallel -sheet. The mutual orientation of the two subdomains was poorly defined by the NMR data. Heteronuclear relaxation data suggested that this disorder resulted from a relative lack of experimental restraints due to the limited size of the interface, rather than inherent high-frequency flexibility. Refinement of the structure using ¹H^N-¹⁵N residual dipolar coupling restraints results in significantly improved definition of the relative subdomain orientations.     

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution

Recombinant Desulfovibrio vulgaris flavodoxin was produced inEscherichia coli. A complete backbone NMR assignment for the two-electronreduced protein revealed significant changes of chemical shift valuescompared to the oxidized protein, in particular for the flavinemononucleotide (FMN)-binding site. A comparison of homo- and heteronuclearNOESY spectra for the two redox states led to the assumption that reductionis not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgarisflavodoxin were investigated using two-dimensional¹⁵N-¹H correlation NMR spectroscopy.T₁, T₂ and NOE data are obtained for 95%of the backbone amide groups in both redox states. These values wereanalysed in terms of the model-free approach introduced by Lipari andSzabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. Acomparison of the two redox states indicates that in the reduced speciessignificantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H groupof FMN bound to the reduced protein compared to the oxidized state.     

A hybrid NMR/SAXS‐based approach for discriminating oligomeric protein interfaces using Rosetta

Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology‐based methods, protein–protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet‐V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution. Proteins 2015; 83:309–317. © 2014 Wiley Periodicals, Inc.     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.     

Solution Structure,Copper Binding and Backbone Dynamics of Recombinant Ber e 1–The Major Allergen from Brazil Nut

Background

The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners.

Methodology/Principal Findings

The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using ¹⁵N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu²⁺ in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale.

Conclusions/Significance

The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu²⁺ ions. This Cu²⁺ binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu²⁺ can in turn affect the interaction that is essential to provoke an allergenic response.