Conformational studies of the C-terminal 16-amino-acid-residue fragment of the B3 domain of the immunoglobulin binding protein G from Streptococcus

The structure and stability of the 16-amino-acid-residue fragment [IG(46-61)] corresponding to the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus was investigated by means of CD and NMR spectroscopy and by differential scanning calorimetry. The CD and 2D NMR experiments were carried out (i) in water at different temperatures and (ii) at one temperature (305 K), with only CD, at different TFE concentrations. Our results show that the IG(46-61) peptide possesses organized three-dimensional structure at all investigated temperatures. The three-dimensional structure of the IG(46-61) peptide resembles the general shape of a beta-hairpin that is also observed for this peptide in the experimental structure of the B3 domain in the whole G protein; the structure is stabilized by hydrophobic interactions between nonpolar side chains. Our study shows that the melting temperature of the IG(46-61) peptide is about 320 K which supports the hypothesis that the investigated peptide can serve as a folding initiation site of the B3 domain of the immunoglobulin binding protein G.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. III. Dynamics of long‐range hydrophobic interactions

A 20‐residue peptide, IG(42–61), derived from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using circular dichroism, nuclear magnetic resonance (NMR) spectroscopy at various temperatures and by differential scanning calorimetry (DSC). Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07 mM), which suggests a three‐state folding/unfolding process. The results from DSC and NMR measurements suggest the formation of a dynamic network of hydrophobic interactions stabilizing the structure, which resembles a β‐hairpin shape over a wide range of temperatures (283–313 K). Our results show that IG (42–61) possesses a well‐organized three‐dimensional structure stabilized by long‐range hydrophobic interactions (Tyr50 ··· Phe57 and Trp48 ··· Val59) at T = 283 K and (Trp48 ··· Val59) at 305 and 313 K. The mechanism of β‐hairpin folding and unfolding, as well as the influence of peptide length on its conformational properties, are also discussed. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. I. Importance of hydrophobic interactions in stabilization of β‐hairpin structure

We previously studied a 16‐amino acid‐residue fragment of the C‐terminal β‐hairpin of the B3 domain (residues 46–61), [IG(46–61)] of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG (46–61) by systematically shortening the peptide by one residue at a time from both the C‐ and the N‐terminus. To determine the structure and stability of two resulting 12‐ and 14‐amino acid‐residue peptides, IG(48–59) and IG(47–60), respectively, we carried out circular dichroism, NMR, and calorimetric studies of these peptides in pure water. Our results show that IG(48–59) possesses organized three‐dimensional structure stabilized by hydrophobic interactions (Tyr50–Phe57 and Trp48–Val59) at T = 283 and 305 K. At T = 313 K, the structure breaks down because of increased chain entropy, but the turn region is preserved in the same position observed for the structure of the whole protein. The breakdown of structure occurs near the melting temperature of this peptide (T_m = 310 K) measured by differential scanning calorimetry (DSC). The melting temperature of IG(47–60) determined by DSC is T_m = 330 K and its structure is similar to that of the native β‐hairpin at all (lower) temperatures examined (283–313 K). Both of these truncated sequences are conserved in all known amino acid sequences of the B domains of the immunoglobulin binding protein G from bacteria. Thus, this study contributes to an understanding of the mechanism of folding of this whole family of proteins, and provides information about the mechanism of formation and stabilization of a β‐hairpin structural element. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. II. Interplay of local backbone conformational dynamics and long‐range hydrophobic interactions in hairpin formation

Two peptides, corresponding to the turn region of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus, consisting of residues 51–56 [IG(51–56)] and 50–57 [IG(50–57)], respectively, were studied by circular dichroism and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β‐turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to those observed in the native protein. The formation of a turn is observed for both peptides in a broad range of temperatures (T = 283–323 K), which confirms the conclusion drawn from our previous studies of longer sequences from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G (16, 14, and 12 residues), that the DDATKT sequence forms a nucleation site for formation of the β‐hairpin structure of peptides corresponding to the C‐terminal part of all the B domains of the immunoglobulin binding protein G. We also show and discuss the role of long‐range hydrophobic interactions as well as local conformational properties of polypeptide chains in the mechanism of formation of the β‐hairpin structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Dissection of the protein G B1 domain binding site for human IgG Fc fragment.

The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine-scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well-defined "knobs-into-holes" interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar "hot spot."     

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.     

Synthesis, folding, and structure of the beta-turn mimic modified B1 domain of streptococcal protein G

The mechanism of beta-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied beta-turn mimic, designated as the dibenzofuran-based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N-terminal beta-hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147-150). The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the beta-turn mimic from acting as a strong beta-sheet nucleator, which reinforces the idea that the native beta-hairpin formation is not driven by the beta-turn formation, but by tertiary interactions.     

Design and structural thermodynamic studies of the chimeric protein derived from spectrin SH3 domain

A number of the chimeric constructs with spectrin SH3 domain were designed for structural and thermodynamic studies of protein self-assembly and protein-ligand interactions. SH3 domains, components of many regulatory proteins, operate through weak interactions with proline-rich regions of polypeptide chains. The recombinant construct (WT-CIIA) studied in this work was constructed by linking the peptide ligand PPPVPPYSAG to the domain C-terminus via a long 12-residue linker to increase the affinity of this ligand for the spectrin domain, thereby ensuring a stable positioning of the polyproline helix to the conserved ligand-binding site in orientation II, which is regarded as untypical of the interaction between this domain and oligopeptides. A comparison of fluorescence spectra of the initial domain and the recombinant protein WT-CIIA suggests that the ligand sticks to the conservative binding site. However, analysis of the equilibrium urea-induced unfolding has demonstrated that this is an unstable contact, which leads to a two-stage unfolding of the chimeric protein. The protein WT-CIIA was crystallized; a set of X-ray diffraction data with a resolution of 1.75 Å was recorded from individual crystals. A preliminary analysis of these diffraction data has demonstrated that the crystals belong to space group P32 with the following unit cell parameters: a = b = 36.39, c = 112.17 Å, a = β = 90.0, and γ = 120.0.     

Study of protein-protein interactions by fluorescence of tryptophan analogs: application to immunoglobulin G binding domain of streptococcal protein G

The protein-protein interaction system often contains many fluorophores that may significantly interfere with the quantitative determination of the binding abilities. To solve this perplexing problem, we biosynthetically incorporated the two tryptophan analogs, 5-hydroxytryptophan and 7-azatryptophan, into the immunoglobulin G (IgG) binding domain of streptococcal protein G (PGBD). The exclusive excitation and novel fluorescence changes in both the intensity and anisotropy are beneficial to reporting the details of the interactions between PGBD and the IgG fragments and enable assessment of the binding abilities. The dissociation constants are estimated to be 0.28 microM for the binding of human Fc and 8.0 microM for mouse Fc. The results clearly demonstrate that labeling of tryptophan analogs has very little effect on the binding abilities and is broadly applicable to quantitatively studying protein-protein interactions in a whole biomolecular complex.     

颗粒裂解肽G13结构域的重组表达及蛋白质结构预测

基因工程构建表达是获得抗菌肽的一种成本较低的方法,本实验人工合成G13结构域编码DNA序列,PCR扩增后,用T-A克隆法与pBAD/TOPO ThioFusion表达载体连接,通过PCR鉴定筛选出正确重组质粒,在大肠杆菌Top10中对目的蛋白进行表达,大肠杆菌工程菌经阿拉伯糖诱导后取样,用SDS-PAGE检测表达情况,采用生物信息学方法对表达蛋白的结构特征进行模拟分析。结果显示：目的蛋白在原核系统中实现了高效表达,表达量高达67%以上,主要以包涵体形式表达。蛋白结构预测结果显示,目的蛋白原有的α螺旋活性结构无改变,从而为抗菌肽高效生产提供了有效可靠的研究途径。     

Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy.

The structure and dynamics of the urea-denatured B1 immunoglobulin binding domain of streptococcal protein G (GB1) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete 1H, 15N, and 13C assignments are obtained by means of sequential through-bond correlations. The nuclear Overhauser enhancement, chemical shift, and 3JHN alpha coupling constant data provide no evidence for the existence of any significant population of residual native or nonnative ordered structure. 15N relaxation measurements at 500 and 600 MHz, however, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second beta-hairpin, the N-terminus of the alpha-helix, and the middle of the alpha-helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (approximately 3 ns) and could range from about 6 ns in the case of one model to between 4 microseconds and 2 ms in another; it is not possible to distinguish between these two cases with certainty because the dynamics are highly complex and hence the analysis of the time scale of this slower motion is highly model dependent. It is suggested that these three regions may correspond to nucleation sites for the folding of the GB1 domain. With the exception of the N- and C-termini, where end effects predominate, the amplitude of the subnanosecond motions, on the other hand, are fairly uniform and model independent, with an overall order parameter S2 ranging from 0.4 to 0.5.     

Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.     

Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the beta-sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13CH2D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.     

The high resolution NMR structure of the third SH3 domain of CD2AP

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of ¹HN, ¹⁵N, ¹Hα, ¹³Cα, ¹³CO and ¹³Cβ chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.     

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.     

Mutations in the B1 domain of protein G that delay the onset of amyloid fibril formation in vitro

We previously reported that under certain experimental conditions, many variants of the B1 domain of IgG-binding protein G from Streptococcus form fibrils reproducibly. The variant I6T53 was the focus of the present study because the lag phase in the kinetics of fibril formation by this variant is significantly longer than that of other variants. This lag phase is distinguished by changes in both intrinsic fluorescence intensity and in light scattering of the protein. NMR diffusion measurements suggest that the soluble protein during the lag phase is monomeric. The kinetic profiles of fibril formation are found to depend on experimental conditions. The first kinetic phase diminishes almost completely when the reaction is seeded with preformed amyloid fibrils.     

The role of backbone conformational heat capacity in protein stability: temperature dependent dynamics of the B1 domain of Streptococcal protein G

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15N NMR-derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross-relaxation rates (eta(xy)), and steady state [1H]-15N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 degrees C for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/eta(xy) was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter (S2) was used to calculate the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temperature (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to approximately 0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be approximately 9 degrees C lower (78 degrees C rather than 87 degrees C). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.