共查询到20条相似文献,搜索用时 15 毫秒
1.
Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional VH:VL antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies. 相似文献
2.
3.
Saerens D 《World journal of biological chemistry》2010,1(7):235-238
It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from all other species, these special antibodies are devoid of light chains, and are composed of a heavy chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol. These HCAbs are easily purified from serum, and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. The antigen binding site of the dromedary HCAb comprises one single domain, referred to as VHH or nanobody (Nb), therefore, a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens. The monoclonal Nb is produced well in bacteria, is very stable and highly soluble, and it binds the antigen with high affinity and specificity. Currently, the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors, to diagnose infections, or to treat diseases such as cancer or trypanosomiasis. 相似文献
4.
Sudhir Paul 《Molecular biotechnology》1996,5(3):197-207
Catalytic activity can arise by natural means in antibodies. Several naturally-occurring peptides and synthetic protease substrates
are known to be cleaved by antibodies. There is an increased production of antigen-specific catalytic antibodies in autoimmune
disease. Antibody light chains isolated from multiple myeloma patients frequently express proteolytic activity. Immunization
protocols using as antigens the ground state of a naturally-occurring polypeptide, transition state analogs or anti-enzyme
antibodies are known to provoke catalytic antibody synthesis. Active site residues in the light chain subunit serve as the
catalytic residues in an antibody with peptide bond cleaving activity. Mutagenesis in the active site can potentially generate
improved catalysts. The possible mechanisms underlying proteolysis by natural antibodies and evolution of the catalytic activity
are reviewed. 相似文献
5.
《MABS-AUSTIN》2013,5(1):219-235
We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (Tm), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their Tms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin. 相似文献
6.
Druar C Saini SS Cossitt MA Yu F Qiu X Geisbert TW Jones S Jahrling PB Stewart DI Wiersma EJ 《Immunogenetics》2005,57(10):730-738
The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire
is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific
primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human VH repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the VH3 (41%), VH4 (39%), and VH1 (14%) subgroups used more frequently than the VH5 (3.9%) or VH7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative
homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding
human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human VH genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution
of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could
be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans. 相似文献
7.
Tereshko V Uysal S Koide A Margalef K Koide S Kossiakoff AA 《Protein science : a publication of the Protein Society》2008,17(7):1175-1187
A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V(H)H) as a crystallization chaperone. By using a yeast surface display system for V(H)H, we successfully introduced additional Met residues in the core of the V(H)H scaffold. We identified a set of SeMet-labeled V(H)H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V(H)H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and V(H)H model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V(H)H, the enhanced V(H)H domain described here will be an excellent scaffold for producing effective crystallization chaperones. 相似文献
8.
Dae Young Kim Rebecca To Hiba Kandalaft Wen Ding Henk van Faassen Yan Luo Joseph D Schrag Nadereh St-Amant Mary Hefford Tomoko Hirama John F Kelly Roger MacKenzie Jamshid Tanha 《MABS-AUSTIN》2014,6(1):219-235
We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (Tm), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their Tms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin. 相似文献
9.
Acierno JP Braden BC Klinke S Goldbaum FA Cauerhff A 《Journal of molecular biology》2007,374(1):130-146
The somatic mutations accumulated in variable and framework regions of antibodies produce structural changes that increase the affinity towards the antigen. This implies conformational and non covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the VH-VL interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. The structure of the free form of the Fab F10.6.6 presented here allows a comparative study of the conformational changes produced upon binding to antigen. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the VH-VL interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen. 相似文献
10.
By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells. 相似文献
11.
Thomas Spreter Von Kreudenstein Eric Escobar-Carbrera Paula I. Lario Igor D’Angelo Karine Brault John F Kelly Yves Durocher Jason Baardsnes R. Jeremy Woods Michael Hongwei Xie Pierre-Alain Girod Michael D. L. Suits Martin J. Boulanger David K. Y. Poon Gordon Y. Ng Surjit B. Dixit 《MABS-AUSTIN》2013,5(5):646-654
While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic. 相似文献
12.
Anita Bosak Bojana Barlović-Tušek Elsa Reiner 《Journal of enzyme inhibition and medicinal chemistry》2013,28(4):521-525
The aim of this study was to differentiate the EDTA-sensitive from the EDTA-insensitive human serum esterases by evaluating their catalytic constants, KM and Vm, for the hydrolysis of phenylacetate (PA). Measurements were done at 37°C in 0.1 M Tris/HCl buffer pH 7.4 and 8.4. The KM,sen and KM,ins constants were significantly different, 0.97 and 2.7 mM respectively, confirming that two esterases hydrolyse PA. The pH of the medium had no effect on KM values, and also no effect on Vm,sen while Vm,ins was two fold higher at pH 8.4 than at 7.4 further confirming the existence of two different enzymes. The stability of the esterases in aqueous media was also studied. EDTA-sensitive activity in buffer without CaCl2 was extremely unstable; the time-course of inactivation followed a two-phase reaction kinetics, indicating that two EDTA-sensitive esterases hydrolyse PA. The EDTA-insensitive activity remained constant in aqueous media under the same experimental conditions. 相似文献
13.
Norihiro Kobayashi Hiroyuki Oyama Tatsuaki Kanda Yoshinori Kato Tadashi Nishio Junichi Goto 《Analytical biochemistry》2009,387(2):257-266
Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17β [E2], cortisol, and 17α-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E2-biotin conjugate was used to generate anti-E2 single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the VH domain of a mouse anti-E2 antibody, and these products were expressed as phagemid particles that were reacted with the E2-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties. 相似文献
14.
Ribosome display is a powerful approach for affinity and stability maturation of recombinant antibodies. However, since ribosome display is performed entirely in vitro, there are several limitations to this approach including technical challenges associated with: (i) efficiently expressing and stalling antibodies on ribosomes using cell-free translation mixtures; and (ii) folding of antibodies in buffers where the concentration and composition of factors varies from that found in the intracellular milieu. We have developed a novel method for intracellular ribosome display that takes advantage of the recently discovered Escherichia coli SecM translation arrest mechanism. Specifically, we provide the first evidence that the encoding mRNA of SecM-stalled heterologous proteins remains stably attached to ribosomes, thereby enabling creation of stalled antibody-ribosome-mRNA (ARM) complexes entirely inside of living cells. Since ARM complexes faithfully maintain a genotype-phenotype link between the arrested antibody and its encoding mRNA, we demonstrate that this method is ideally suited for isolating stability-enhanced single-chain variable fragment (scFv) antibodies that are efficiently folded and functional in the bacterial cytoplasm. 相似文献
15.
Peter Bartels Kerstin Behnke Guido Michels Ferdi Groner Toni Schneider Margit Henry Paula Q. Barrett Ho-Won Kang Jung-Ha Lee Martin H.J. Wiesen Jan Matthes Stefan Herzig 《Cell calcium》2009,46(4):293-302
We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels CaV3.1 and CaV3.2 by nitrous oxide (N2O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N2O significantly inhibited CaV3.2, and – less pronounced – CaV3.1. A CaV3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N2O reduced average peak current of CaV3.2 by decreasing open probability and open time duration. Effects on CaV3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single CaV3.1 channels were significantly less active than CaV3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within CaV3.2 (H191), are responsible for the subtype-prevalent N2O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N2O on CaV3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant CaV3.1. 相似文献
16.
Alexander A. Kortt Robin E. Guthrie Mark G. Hinds Barbara E. Power Neva Ivancic J. Bruce Caldwell L. Clem Gruen Raymond S. Norton Peter J. Hudson 《Journal of Protein Chemistry》1995,14(3):167-178
The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAGTM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its1H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained. 相似文献
17.
McAuley A Jacob J Kolvenbach CG Westland K Lee HJ Brych SR Rehder D Kleemann GR Brems DN Matsumura M 《Protein science : a publication of the Protein Society》2008,17(1):95-106
Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody. 相似文献
18.
For decades, the binding of prostaglandin H2 (PGH2) to multiple target proteins of unrelated protein structures which mediate diverse biological functions has remained a real mystery in the field of eicosanoid biology. Here, we report that the structure of a PGH2 mimic, U46619, bound to the purified human TP, was determined and compared with that of its conformation bound to the COX-downstream synthases, prostacyclin synthase (PGIS) and thromboxane A2 synthase (TXAS). Active human TP protein, glycosylated and in full length, was expressed in Sf-9 cells using a baculovirus (BV) expression system and then purified to near homogeneity. The binding of U46619 to the purified receptor in a nonionic detergent-mimicked lipid environment was characterized by high-resolution NMR spectroscopy. The conformational change of U46619, upon binding to the active TP, was evidenced by the significant perturbation of the chemical shifts of its protons at H3 and H4 in a concentration-dependent manner. The detailed conformational changes and 3D structure of U46619 from the free form to the TP-bound form were further solved by 2D 1H NMR experiments using a transferred NOE (trNOE) technique. The distances between the protons of H11 and H18, H11 and H19, H15 and H18, and H15 and H19 in U46619 were shorter following their binding to the TP in solution, down to within 5 Å, which were different than that of the U46619 bound to PGIS and U44069 (another PGH2 mimic) bound to TXAS. These shorter distances led to further separation of the U46619 α and ω chains, forming a unique “rectangular” shape. This enabled the molecule to fit into the ligand-binding site pocket of a TP model, in which homology modeling was used for the transmembrane (TM) domain, and NMR structures were used for the extramembrane loops. The proton perturbations and 3D conformations in the TP-bound U46619 were different with that of the PGH2 mimics bound to PGIS and TXAS. The studies indicated that PGH2 can adopt multiple conformations in solution to satisfy the specific and unique shapes to fit the different binding pockets in the TP receptor and COX-downstream enzymes. The results also provided sufficient information for speculating the molecular basis of how PGH2 binds to multiple target proteins even though unrelated in their protein sequences. 相似文献
19.
It has been demonstrated in several insect species that a circadian clock makes the whole of antennal chemoreceptors more sensitive during a particular temporal window every day. This assessment raises the question about how insects exhibiting bimodal activity handle their sensitivity to odours which are relevant at different moments of the day. To shed some light on this problem, we studied in Rhodnius prolixus the daily dynamics of their responsiveness to CO2 (host-associated cue) and aggregation cues (refuge-associated), which are relevant at dusk and dawn, respectively. We analysed: (1) whether a temporal modulation of the responsiveness to odours does exist in R. prolixus, (2) if this modulation is a general one or it is specific for each type of volatile, and (3) if it is controlled by exogenous or endogenous mechanisms. We found that the responsiveness to CO2 only occurs at dusk and that to assembling odours is restricted to dawn. Experiments under free-running conditions revealed that only the responsiveness to CO2 is controlled by a circadian clock, but not that to assembling signals. Thus, by combining endogenous and exogenous mechanisms, sensitivities to different odours are adjusted according to their associated behavioural context and moment of the day. 相似文献
20.
Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold. 相似文献