Solvent-exposed Amino Acid Residues Research Articles

Dynamics of proteins with different molecular structures under solution condition

Incoherent quasielastic neutron scattering (iQENS) is a fascinating technique for investigating the internal dynamics of protein. However, low flux of neutron beam, low signal to noise ratio of QENS spectrometers and unavailability of well-established analyzing method have been obstacles for studying internal dynamics under physiological condition (in solution). The recent progress of neutron source and spectrometer provide the fine iQENS profile with high statistics and as well the progress of computational technique enable us to quantitatively reveal the internal dynamic from the obtained iQENS profile. The internal dynamics of two proteins, globular domain protein (GDP) and intrinsically disordered protein (IDP) in solution, were measured with the state-of-the art QENS spectrometer and then revealed with the newly developed analyzing method. It was clarified that the average relaxation rate of IDP was larger than that of GDP and the fraction of mobile H atoms of IDP was also much higher than that of GDP. Combined with the structural analysis and the calculation of solvent accessible surface area of amino acid residue, it was concluded that the internal dynamics were related to the highly solvent exposed amino acid residues depending upon protein’s structure.

Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization.

Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure. The methodology relies on "painting" the reactive and solvent-exposed amino acid residues with chemical tags and using the pattern of modifications as footprints from analysis by bottom-up MS-based proteomics to deduce protein higher order structures. The outcome can refer to proteins in solution or even in cells and is complementary to those of X-ray crystallography and NMR. It is particularly useful in mapping protein-ligand interfaces and conformational changes resulting from ligand binding, mutation, and aggregation. Fast photochemical oxidation of proteins (FPOP), in its original conception, is a type of hydroxyl-radical-based protein footprinting that utilizes a pulsed KrF laser (248 nm) to trigger hydrolysis of hydrogen peroxide to produce solution hydroxyl radicals, which subsequently modify the protein in situ. The platform is expanding to adopt other reactive species including carbenes. The reactivity of the probe depends on the intrinsic reactivity of the radical with the residue side chain and the solvent accessibility of the residue as a function of the tertiary/quaternary structures. By introducing an appropriate scavenger to compete with hydroxyl radical self-quenching, the lifetime of the primary radicals is remarkably shortened to approximately microsecond. Thus, the sampling time scale of FPOP is much faster than hydrogen-deuterium exchange and other covalent labeling methods relying on nonradical reactions. The short footprinting time scale of FPOP offers two major advantages for protein structure elucidation: (1) it allows the protein to be interrogated in its native or near-native state with minimum structural perturbation; (2) it exhibits high sensitivity toward alterations in protein higher order structures because its sampling time is short with respect to protein conformational changes and dynamic motion. In addition, the covalent and irreversible oxidation by the hydroxyl radical provides more flexibility in the downstream proteomics workflow and MS analysis, permitting high spatial resolution with residue-specific information. Since its invention in 2005 by Hambly and Gross, FPOP has developed from proof-of-concept to a valuable biophysical tool for interrogating protein structure. In this Account, we summarize the principles and experimental design of FPOP that enable its fast labeling and describe the current and unique capabilities of the technique in protein higher order structure elucidation. Application examples include characterization of amyloid β self-assembly, protein-ligand interactions with a special emphasis on epitope mapping for protein therapeutics (e.g., antibody, Fab, and adnectin), protein folding detailed to residue-specific folding kinetics, and protein flexibility/dynamics. Additionally, the utility of FPOP-based oxidative footprinting should grow with our continuing developments of novel reagents (e.g., sulfate radical anion, carbene diradical, and trifluoromethyl radical). These reactive reagents are compatible with the current FPOP platform and offer different reactivity and selectivity toward various types of amino acid residues, providing complementary insights into protein higher order structures for soluble proteins and ultimately for membrane-bound proteins.

Fast internal dynamics in alcohol dehydrogenase.

Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

Cleavable ester linked magnetic nanoparticles for labeling of solvent exposed primary amine groups of peptides/proteins.

Covalent labeling of solvent exposed amino acid residues using chemical reagents/crosslinkers followed by mass spectrometric analysis can be used to determine the solvent accessible amino acids of a protein. A variety of chemical reagents containing cleavable bonds were developed to label abundantly found lysine residues on the surface of protein. To achieve efficient separation of labeled peptides prior to mass spectrometric analysis, magnetic nanoparticles can be decorated with amino acid reactive functional groups and utilized for quick recovery of labeled peptides. [1] In this work, iron oxide magnetic nanoparticles (Fe3O4 MNPs) were synthesized by thermal decomposition method and coated with silica (SiO2@Fe3O4 MNPs) by reverse micro emulsion approach. The Fe3O4 MNPs and SiO2@Fe3O4 MNPs were characterized by TEM and XRD. The SiO2@Fe3O4 MNPs were further coated with amine groups and conjugated to N-hydroxysuccinimidyl (NHS) ester groups via a cleavable ester bond. Fluorescence based qualitative analysis of ester linked NHS ester modified SiO2@Fe3O4 MNPs was performed to confirm the presence of NHS ester group. The active NHS ester sites on the surface of SiO2@Fe3O4 MNPs were determined by depletion approach and found to be 694 active sites per 1 mg of SiO2@Fe3O4 MNPs. Free amine groups of a small peptide, ACTH (4–11) were labeled by ester linked, NHS ester modified SiO2@Fe3O4 MNPs under physiological conditions. Superparamagnetic nature of SiO2@Fe3O4 MNPs allowed quick and efficient magnetic separation of labeled peptides from the solution. The ester bond was further cleaved to separate labeled peptides followed by mass spectrometric analysis. The ester linked, NHS ester modified SiO2@Fe3O4 MNPs introduced a mass shift of 115.09 Da on amine groups of ACTH (4–11), which was confirmed by mass spectrometry.

Halogenation of β-estradiol by a rationally designed mesoporous biocatalyst based on chloroperoxidase

AbstractChloroperoxidase from Caldariomyces fumago was immobilized in Eupergit® C, a commercial mesoporous acrylic-based material. Due to low stability of the enzyme under neutral and basic pH, the usual covalent immobilization procedures cannot be applied to this enzyme. Several strategies were followed in order to achieve a stable interaction between the protein and the support. The support was efficiently functionalized with different reactive groups such as aromatic and aliphatic amines, glutaraldehyde, diazonium ions, and maleimide moieties; solvent-exposed amino acid residues in chloroperoxidase were identified or created through chemical modification, so that they were reactive under conditions where the enzyme is stable. Enzyme load and retained activity were monitored, obtaining biocatalysts with specific activity ranging from 200 to 25,000 U/g. The highest load and activity was obtained from the immobilization of a chemically-modified CPO preparation bearing a solvent-exposed free thiol group. This biocatalyst efficiently catalyzed the transformation of β-estradiol, an endocrine disruptor.

Interactions of cyclodextrins with aromatic amino acids: a basis for protein interactions

Cyclodextrins (CyD) have proven effects on the stability of proteins and can be used in the formulation of aggregation prone therapeutic proteins. This effect stems from specific interactions between the CyD (preferably β-CyD) and solvent exposed amino acid residues. Here the interaction with hydrophobic aromatic amino acid residues stands out and the interaction between CyDs and these amino acid residues holds the key to understanding the observed effects, which CyDs exerts on proteins and peptides. Here we present a comparative study of the interactions between free and peptide bound aromatic amino acids and their derivatives with α, β and γ-CyDs using NMR spectroscopy. We propose a novel, quantitative means of assessing the penetration depth of guest molecules in CyD cavities, the penetration gauge Π, and apply it to the observed interaction patterns from ROESY NMR spectra. We demonstrate that the penetration depths of the aromatic rings within the CyDs rely highly on the nature of the remainder of the guest molecule. Thus the presence of charges, neighboring amino acids and the specific positioning on the surface of a protein highly influences the penetration depth and geometry of guest–CyD interactions.

Affilin–Novel Binding Molecules Based on Human γ-B-Crystallin, an All β-Sheet Protein

The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a β-sheet of human γ-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type γ-B-crystallin and a selected Affilin variant have been determined to 1.7 Å and 2.0 Å resolution, respectively. Comparison of the two molecules indicates that the human γ-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed γ-B-crystallin provides a suitable framework for the generation of novel binding molecules.

Xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as Xa26

Xa3-mediated resistance for rice bacterial blight, one of the most devastating rice diseases worldwide, is influenced by genetic background. Xa3 is genetically tightly linked to Xa26, another gene for bacterial blight resistance. Xa26 belongs to a clustered multigene family encoding leucine-rich repeat (LRR) receptor kinase-like proteins. To characterize Xa3, we fine mapped it using a population segregating for only one resistance gene and markers developed from Xa26 family. Genetic analysis showed that Xa3 co-segregated with the marker of Xa26 gene and segregated from the markers of other members of Xa26 family. DNA fingerprinting revealed that rice line IRBB3 carrying Xa3 had the same copy numbers of Xa26 family members as rice line Minghui 63 carrying Xa26. Phenotypic comparison showed that all the rice lines carrying either Xa3 or Xa26 developed dark brown deposition at the border between the lesion caused by incompatible-pathogen infection and health leaf tissue, while other rice lines did not show this dark brown deposition in either incompatible or compatible interactions. These results suggest that Xa3 and Xa26 is the same gene. We name it Xa3/Xa26 to indicate the relationship between the two gene symbols. The putative encoding products of Xa3/Xa26 and its susceptible allele xa3/xa26 shared 92% sequence identity. The sequence difference occurred in the LRR domains, specifically at the solvent-exposed amino acid residues, might be the major cause that differentiates the resistant and susceptible proteins.

Point Mutations with Positive Selection Were a Major Force during the Evolution of a Receptor-Kinase Resistance Gene Family of Rice

The rice (Oryza sativa) Xa26 gene, which confers resistance to bacterial blight disease and encodes a leucine-rich repeat (LRR) receptor kinase, resides at a locus clustered with tandem homologous genes. To investigate the evolution of this family, four haplotypes from the two subspecies of rice, indica and japonica, were analyzed. Comparative sequence analysis of 34 genes of 10 types of paralogs of the family revealed haplotype polymorphisms and pronounced paralog diversity. The orthologs in different haplotypes were more similar than the paralogs in the same haplotype. At least five types of paralogs were formed before the separation of indica and japonica subspecies. Only 7% of amino acid sites were detected to be under positive selection, which occurred in the extracytoplasmic domain. Approximately 74% of the positively selected sites were solvent-exposed amino acid residues of the LRR domain that have been proposed to be involved in pathogen recognition, and 73% of the hypervariable sites detected in the LRR domain were subject to positive selection. The family is formed by tandem duplication followed by diversification through recombination, deletion, and point mutation. Most variation among genes in the family is caused by point mutations and positive selection.

Solvent Exposed Non-contacting Amino Acids Play a Critical Role in NF-κB/IκBα Complex Formation

IκBα inhibits transcription factor NF-κB activity by specific binding to NF-κB heterodimers composed of p65 and p50 subunits. It binds with slightly lower affinity to p65 homodimers and with significantly lower affinity to homodimers of p50. We have employed a structure-based mutagenesis approach coupled with protein–protein interaction assays to determine the source of this dimer selectivity exhibited by IκBα. Mutation of amino acid residues in IκBα that contact NF-κB only marginally affects complex binding affinity, indicating a lack of hot spots in NF-κB/IκBα complex formation. Conversion of the weak binding NF-κB p50 homodimer into a high affinity binding partner of IκBα requires transfer of both the NLS polypeptide and amino acid residues Asn202 and Ser203 from the NF-κB p65 subunit. Involvement of Asn202 and Ser203 in complex formation is surprising as these amino acid residues occupy solvent exposed positions at a distance of 20 Å from IκBα in the crystal structures. However, the same amino acid residue positions have been genetically isolated as determinants of binding specificity in a homologous system in Drosophila. X-ray crystallographic and solvent accessibility experiments suggest that these solvent-exposed amino acid residues contribute to NF-κB/IκBα complex formation by modulating the NF-κB p65 subunit NLS polypeptide.

Evolutionary dynamics of plant R-genes.

Plant R-genes involved in gene-for-gene interactions with pathogens are expected to undergo coevolutionary arms races in which plant specificity and pathogen virulence continually adapt in response to each other. Lending support to this idea, the solvent-exposed amino acid residues of leucine-rich repeats, a region of R-genes involved in recognizing pathogens, often evolve at unusually fast rates. But within-species polymorphism is also common in R-genes, implying that the adaptive substitution process is not simply one of successive selective sweeps. Here we document these features in available data and discuss them in light of the evolutionary dynamics they likely reflect.

Probing the Escherichia coli transcriptional activator MarA using alanine-scanning mutagenesis: residues important for DNA binding and activation

The MarA transcriptional activator binds to a 20 bp asymmetric degenerate sequence (marbox) located at different positions and orientations within the promoters of the genes of the Escherichia coli mar regulon. Solution of the MarA-marbox X-ray crystallographic structure suggested the presence of base-specific and non-specific interactions between the marbox and two helix-turn-helix (HTH) motifs on the monomeric MarA. Here, we use alanine-scanning mutagenesis and DNA retardation analysis to: (i) evaluate the contacts between MarA and the marboxes of five differently configured mar regulon promoters; (ii) assess the role of conserved hydrophobic amino acid residues for MarA activity; and (iii) identify residues required for RNA polymerase activation. These analyses revealed that the phosphate-backbone contacts and hydrogen bonds with the bases of the marbox are more significant for DNA binding than are the van der Waals interactions. While both N and C-terminal HTH motifs make essential contributions to binding site affinity, MarA is more sensitive to alterations in the N-terminal HTH. In a similar way, the activity of MarA is more sensitive to alterations in the hydrophobic core of this HTH. Solvent-exposed amino acid residues located at many positions on the MarA surface are important for activity. Some of these residues affect activity on all promoters and thus, are implicated in maintaining MarA structure whereas several solvent-exposed amino acids not involved in DNA binding were important for MarA activity on specific promoters. The pattern of activation defects defined a class II promoter-specific activating region. However, a localized class I activating region was not apparent. These results suggest that MarA activates transcription by at least two distinct mechanisms. Furthermore, the important role of phosphate contacts in marbox affinity suggests that indirect readout contributes to binding site recognition by MarA.

Identification of an immunologically important hypervariable domain of major outer surface protein A of Borrelia burgdorferi.

The gene for the major outer surface protein A (OspA) from several clinically obtained strains of Borrelia burgdorferi, the cause of Lyme disease, has been cloned, sequenced, and expressed in Escherichia coli by using a T7-based expression system (J. J. Dunn, B. N. Lade, and A. G. Barbour, Protein Expr. Purif. 1:159-168, 1990). All of the OspAs have a single conserved tryptophan at residue 216 or, in some cases, 217; however, the region of the protein flanking the tryptophan is hypervariable, as determined by a moving-window population analysis of ospA from 15 European and North American isolates of B. burgdorferi. Epitope-mapping studies using chemically cleaved OspA and a TrpE-OspA fusion have indicated that this hypervariable region is important for immune recognition. Biophysical analysis, including fluorescence and circular dichroism spectroscopy, have indicated that the conserved tryptophan is buried in a hydrophobic environment. Polar amino acid side chains flanking the tryptophan are likely to be exposed to the hydrophilic solvent. The hypervariability of these solvent-exposed amino acid residues may contribute to the antigenic variation in OspA. To test this, we have performed site-directed mutagenesis to replace some of the potentially exposed amino acid side chains in the B31 protein with the analogous residues of a Borrelia garinii strain, K48. The altered proteins were then analyzed by Western blot (immunoblot) with monoclonal antibodies which bind OspA on the surface of the intact B31 spirochete. Our results indicate that specific amino acid changes near the tryptophan can abolish the reactivity of OspA to these monoclonal antibodies, which is an important consideration in the design of vaccines based on recombinant OspA.

Mechanisms of Antibody Binding to a Protein

The mechanisms of antibody binding to a protein were studied by an analysis of specific amino acid residues critical to nine antigenic sites on myohemerythrin. Rabbit antisera to the whole protein were assayed for binding to more than 1500 distinct peptide analogs differing from the protein sequence by single amino acid replacements. The results, combined with information from the three-dimensional crystallographic structure, were used to evaluate probable mechanisms of antibody binding at individual sites. The data from all sites examined indicate that initial binding to solvent-exposed amino acid residues may promote local side-chain displacements and thereby allow the participation of other, previously buried, residues.