Unfolding and refolding of a type kappa immunoglobulin light chain and its variable and constant fragments

By limited proteolysis of a type kappa immunoglobulin light chain (Oku) with clostripain, both the VL and CL fragments were obtained with a high yield. The unfolding and refolding by guanidine hydrochloride of light chain Oku and its VL and CL fragments were studied at pH 7.5 and 25 degrees C with circular dichroism and tryptophyl fluorescence. The concentration of guanidine hydrochloride at the midpoint of the unfolding curve was 1.2 M for the VL fragment and 0.9 M for the CL fragment. As in the case of the CL fragment of light chain Nag (type lambda) [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910], the unfolding and refolding kinetics of the CL fragment could be explained in principle on the basis of the three-species mechanism U1 in equilibrium U2 in equilibrium N, where N is native protein and U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein. The unfolding and refolding kinetics of the VL(Oku) fragment were described by a single exponential term. Double-jump experiments, however, showed that two forms of the unfolding molecule exist. The equilibrium and kinetics of unfolding of light chain Oku were explained by the sum of those of the VL and CL fragments. On the other hand, the refolding kinetics of light chain Oku were greatly different from the sum of those of the VL and CL fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of the buried intrachain disulfide bond of the constant fragment of the immunoglobulin light chain: global unfolding under physiological conditions

The constant (CL) fragment of the immunoglobulin light chain contains only one intrachain disulfide bond buried in the interior of the molecule. The kinetics of reduction with dithiothreitol of the disulfide bond were studied at various concentrations of guanidine hydrochloride at pH 8.0 and 25 degrees C. It was found that the disulfide bond is reduced even in the absence of guanidine hydrochloride. The results of the reduction kinetics were compared with those of the unfolding and refolding kinetics of the CL fragment previously reported [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910]. It was shown that the reduction of the disulfide bond proceeds through a species with a conformation very similar to that of the fully unfolded one and that the CL fragment undergoes global unfolding transition even in water.     

Unfolding and refolding of the reduced constant fragment of the immunoglobulin light chain: Kinetic role of the intrachain disulfide bond

The constant fragment of the immunoglobulin light chain whose intrachain disulfide bond is reduced (reduced C_L fragment) assumes a conformation very similar to the intact C_L fragment (Goto & Hamaguchi, 1979). The kinetics of reversible unfolding and refolding of the reduced C_L fragment by guanidine hydrochloride at pH 7.5 and 25 °C were studied and were compared with those of the intact C_L fragment described in the accompanying paper (Goto & Hamaguchi, 1982). Tryptophyl fluorescence, far-ultraviolet circular dichroism, and reactivity of the SH groups toward 5,5′-dithiobis-(2-nitrobenzoic acid) were used to follow the kinetics. The results obtained were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The rate constants of interconversion between U₁ and U₂ and the rate constant for the process from N to U₂ were very similar to the respective values for the intact C_L fragment. Only the rate constant for the process from U₂ to N was greatly different between the intact and reduced C_L fragments; the rate constant for the reduced C_L fragment was about 100 times smaller than that for the intact C_L fragment. These results indicated that the slow isomerization of the unfolded molecule is independent of the presence of the disulfide bond and that the kinetic role of the intrachain disulfide bond is to accelerate the folding process. This kinetic role in the folding of the C_L fragment was explainable only in terms of the decreased entropy in the unfolded state of the intact C_L fragment due to the presence of the disulfide bond.     

Role of amino-terminal residues in the folding of the constant fragment of the immunoglobulin light chain

Three constant fragments with different amino terminals, CL(105-214), CL(109-214), and CL(113-214), were obtained by limited proteolysis with trypsin or papain of a type lambda immunoglobulin light chain. The conformations of the three CL fragments were indistinguishable on the basis of circular dichroism and tryptophyl fluorescence spectra. The stability to heat and guanidine hydrochloride of CL(105-214) was almost the same as that of CL(109-214), but the stability of CL(113-214) was slightly lower than that of CL(105-214) or CL(109-214). The midpoint of the thermal unfolding transition at pH 7.5 was at 60.0 degrees C for CL(105-214), 60.4 degrees C for CL(109-214), and 57.5 degrees C for CL(113-214). The midpoint of the unfolding transition by guanidine hydrochloride at pH 7.5 and 25 degrees C was 1.2 M for CL(105-214) and CL(109-214) and at 1.0 M for CL(113-214). The kinetics of unfolding and refolding by guanidine hydrochloride of these CL fragments were analyzed on the basis of the three-species mechanism, U1 in equilibrium with U2 in equilibrium with N, where U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. It was found that only the microscopic unfolding rate constant for CL(113-214) is 2-3 times greater than that for CL(105-214) or CL(109-214) and that the other microscopic rate constants for the three CL fragments are all the same. These findings indicated that the amino-terminal residues, Gly-109-Lys-112, or a part of them, stabilize the CL(113-214) fragment by decreasing only the unfolding rate, that the transition state of the folding of the CL fragment is independent of the presence or absence of this peptide, and that, at the last step of folding, the peptide is incorporated into the globular domain, thus stabilizing it.     

Effects of ammonium sulfate on the unfolding and refolding of the variable and constant fragments of an immunoglobulin light chain

The equilibria and kinetics of unfolding and refolding by guanidine hydrochloride of the VL and CL fragments of a type kappa immunoglobulin light chain were studied in the presence of ammonium sulfate using circular dichroism and tryptophyl fluorescence at pH 7.5 and 25 degrees C. The unfolding equilibria of the VL and CL fragments were described in terms of the two-state transition. The midpoints of unfolding in the absence of ammonium sulfate were at 0.9 and 1.2 M guanidine hydrochloride for the CL and VL fragments respectively. The transition curves were shifted to higher concentrations of guanidine hydrochloride by 1.4 and 1.6 M for the CL and VL fragments, respectively, per mole of ammonium sulfate. Unfolding reactions of the VL and CL fragments in 3 M guanidine hydrochloride followed first-order kinetics, and the rate constants for the two proteins were both greatly decreased by the presence of ammonium sulfate. The refolding reaction of the CL fragment in 0.3 M guanidine hydrochloride consisted of two phases, and the rate constants were increased a little by the presence of ammonium sulfate. The refolding reaction of the VL fragment in 0.3 M guanidine hydrochloride followed first-order kinetics, and the rate was not affected by the presence of ammonium sulfate. These results showed that ammonium sulfate stabilizes the CL and VL fragments mainly by decreasing the unfolding rate.     

Conformation and stability of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge

The constant fragment of the immunoglobulin light chain in which the intramolecular disulfide bond is reduced (reduced CL fragment) assumes a conformation very similar to that of the intact CL fragment and contains two sulfhydryl groups buried in the interior of the molecule [Goto, Y., & Hamaguchi, K. (1979) J. Biochem. (Tokyo) 86, 1433-1441]. In order to understand the role of the disulfide bond, a derivative in which the disulfide bond is replaced by an S-Hg-S bond was prepared and its conformation and stability were studied. The derivative was prepared by reacting the reduced CL fragment with mercuric chloride. Kinetic studies showed that the reaction is rate-limited by the unfolding process of the reduced CL fragment. The mercury derivative was as compact as the intact CL or reduced CL fragment, and a tryptophyl residue was found to be buried near the S-Hg-S bond in the interior of the protein molecule. Judging from the circular dichroic spectrum, however, the beta-structure characteristic of the immunoglobulin fold was disturbed. The stability of the derivative to guanidine hydrochloride was lower than that of the intact CL fragment, but the unfolding transition was reversible and cooperative. Decreased stability of the mercury derivative is due to its folded conformation being distorted by introduction of the S-Hg-S bond.     

Global fluctuations of the immunoglobulin domains under physiological conditions

Hydrogen-exchange rates of the indole NH proton of a tryptophan residue, buried fully in the interior of each of the constant (CL) and variable (VL) fragments of a type-kappa-immunoglobulin light chain, were studied at various pH values and at 25 degrees C under 1H-nuclear magnetic resonance. The activation energies for the exchange reactions were determined also and compared with those for the unfolding reactions of these fragments induced by guanidine hydrochloride. The pH profiles of the exchange rates of the CL(kappa) and VL(kappa) fragments were very similar to that for a CL (lambda) fragment reported previously. It was found that the CL (kappa) and VL (kappa) fragments as well as the CL (lambda) fragment undergo a global unfolding transition with a conformation very similar to that of the fully unfolded state induced by guanidine hydrochloride even under physiological conditions.     

Use of fluorescence energy transfer to characterize the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding

The CL fragment of a type-kappa immunoglobulin light chain in which the C-terminal cysteine residue was modified with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (CL-AEDANS fragment) was prepared. This fragment has only one tryptophan residue at position 148. The compactness of the fragment whose intrachain disulfide bond was reduced in order for the tryptophan residue to fluoresce (reduced CL-AEDANS fragment) was studied in the early stages of refolding from 4 M guanidine hydrochloride by fluorescence energy transfer from Trp 148 to the AEDANS group. The AEDANS group attached to the SH group of a cysteine scarcely fluoresced when excited at 295 nm. For the reduced CL-AEDANS fragment, the fluorescence emission band of the Trp residue overlapped with the absorption band of the AEDANS group, and the fluorescence energy transfer was observed between Trp 148 and the AEDANS group in the absence of guanidine hydrochloride. In 4 M guanidine hydrochloride, the distance between the donor and the acceptor was larger, and the efficiency of the energy transfer became lower. The distance between Trp 148 and the AEDANS group for the intact protein estimated by using the energy-transfer data was in good agreement with that obtained by X-ray crystallographic analysis. By the use of fluorescence energy transfer, tryptophyl fluorescence, and circular dichroism at 218 nm, the kinetics of unfolding and refolding of the reduced fragment were studied. These three methods gave the same unfolding kinetic pattern. However, the refolding kinetics measured by fluorescence energy transfer were different from those measured by tryptophyl fluorescence and circular dichroism, the latter two giving the same kinetic pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain

The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism.The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U₂ to U₁ was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U₁

U₂ reaction is proline isomerization. The rates of interconversion between N and U₂ changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.     

Hydrogen-exchange kinetics of the indole NH proton of the buried tryptophan in the constant fragment of the immunoglobulin light chain

The constant fragment of the immunoglobulin light chain (type lambda) has two tryptophyl residues at positions 150 and 187. Trp-150 is buried in the interior, and Trp-187 lies on the surface of the molecule. The hydrogen-deuterium exchange kinetics of the indole NH proton of Trp-150 were studied at various pH values at 25 degrees C by 1H nuclear magnetic resonance. Exchange rates were approximately first order in hydroxyl ion dependence above pH 8, were relatively independent of pH between pH 7 and 8, and decreased below pH 7. On the assumption that the exchange above pH 8 proceeds through local fluctuations of the protein molecule, the exchange rates between pH 7 and 8 through global unfolding were estimated. The exchange rate constant within this pH range at 25 degrees C thus estimated was consistent with that of the global unfolding of the constant fragment under the same conditions as those reported previously [Kikuchi, H., Goto, Y., & Hamaguchi, K. (1986) Biochemistry 25, 2009-2013]. The activation energy for the exchange process at pH 7.8 was the same as that for the unfolding process by 2 M guanidine hydrochloride. The exchange rates of backbone NH protons were almost the same as that of the indole NH proton of Trp-150 at pH 7.1. These observations also indicated that the exchange between pH 7 and 8 occurs through global unfolding of the protein molecule and is rate-limited by the unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of tryptophan residues and stability changes in proteins

The role of tryptophan residues in the stability of proteins was studied by ozone oxidation, which causes a small change in the tryptophan side chain. Trp 187 of the constant fragment of a type lambda immunoglobulin light chain, Trp 59 of ribonuclease T1, and Trp 62 of hen egg white lysozyme were oxidized specifically by ozone to N'-formylkynurenine or kynurenine. Judging from their circular dichroic and fluorescence spectra, these modified proteins were found to be the same as those of the respective intact proteins. However, even the slight modification of a single tryptophan residue produced a large decrease in the stability of these proteins to guanidine hydrochloride and heat. The smaller the extent of exposure of the tryptophan residue, the greater the effect of the modification on the stability. The formal kinetic mechanism of unfolding and refolding by guanidine hydrochloride of the CL fragment was not altered by tryptophan oxidation, but the rate constants for unfolding and refolding changed. The thermal unfolding transitions were analyzed to obtain the thermodynamic parameters. The enthalpy and entropy changes for the modified proteins were larger than the respective values for the intact proteins.     

Extrinsic 33-kilodalton protein of spinach oxygen-evolving complexes: kinetic studies of folding and disulfide reduction

The 33-kDa protein is one of the three extrinsic proteins in the oxygen-evolving photosystem II complexes. The protein has one intrachain disulfide bond. On reduction of this disulfide bond, the protein was unfolded and lost its activity. On the basis of the unfolding equilibrium curve obtained by using guanidine hydrochloride, the free energy change of unfolding in the absence of guanidine hydrochloride was estimated to be 4.4 kcal/mol using the Tanford method [Tanford, C. (1970) Adv. Protein Chem. 24, 1-95] and 2.8 kcal/mol using the linear extrapolation method. The unfolding of the 33-kDa protein caused by reduction was explained in terms of the entropy change associated with reduction of the intrachain disulfide bond. The kinetics of the reduction of the disulfide bond using dithiothreitol were studied at various concentrations of guanidine hydrochloride at pH 7.5 and 25 degrees C. The disulfide bond was reduced even in the absence of guanidine hydrochloride. The unfolding and refolding kinetics of the 33-kDa protein using guanidine hydrochloride were also studied under the same conditions, and the results were compared with those for the reduction kinetics. It was shown that the reduction of the disulfide bond proceeds through a species in which the disulfide bond is exposed by local fluctuations.     

The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

The nature of the rate-limiting steps in the refolding of the cofactor-dependent protein aspartate aminotransferase

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.     

Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.     

The role of the intrachain disulfide bond in the conformation and stability of the constant fragment of the immunoglobulin light chain.

The conformation and stabilities of the CL fragment isolated from a type lambda Bence Jones protein and the fragment in which the intrachain disulfide bond had been reduced were studied by measuring CD, fluorescence, and ultraviolet absorption. The results indicated that no great conformational change occurs on reduction of the disulfide, unless the SH groups are alkylated. Intact CL was more resistant than reduced CL to guanidine hydrochloride. The denaturation curves were analyzed using an equation based on the binding of guanidine hydrochloride and the free energy changes of denaturation in the absence of the denaturant were estimated as about 6 kcal.mol-1 for intact CL and about 1.8 kcal.mol-1 for reduced CL. The difference in stability between intact CL and reduced CL was explained to a great extent in terms of the entropy change associated with reduction of the intrachain disulfide bond of the fragment in the denatured state.     

Influence of an extrinsic cross-link on the folding pathway of ribonuclease A. Kinetics of folding-unfolding

The kinetics of folding/unfolding of cross-linked Lys7-dinitrophenylene-Lys41-ribonuclease A were studied and compared to those of unmodified ribonuclease A (RNase A) at various concentrations of guanidine hydrochloride. The folding of the denatured cross-linked protein involved one fast-folding species (22 +/- 4%) and two slow-folding species, as observed in unmodified ribonuclease A. Also, a nativelike intermediate, analogous to that reported previously for unmodified ribonuclease A [Cook, K. H., Schmid, F. X., & Baldwin, R. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6157], has been detected on the folding pathway of cross-linked ribonuclease A. The extrinsic cross-link between Lys7 and Lys41 did not affect the rate constants for the folding kinetics of these three species. The cross-link did, however, significantly affect the rate constant for unfolding of the native protein. The conformation of the protein in the transition state of the unfolding pathway was deduced from an analysis of the kinetic data. It appears that the 41 N-terminal residues are unfolded in the transition state of the unfolding pathway. Thus, the unfolding pathway of RNase A is sequential in that further unfolding (after the transition state) follows the unfolding of the 41 N-terminal residues. Also, the conformation of the 41 N-terminal residues does not play a role in the folding pathway. Presumably, if the cross-link were introduced instead between two other residues that are in the segment(s) involved in the rate-limiting step(s), it could increase the refolding rate constants and possibly the concentration of fast-folding species.     

A kinetic trap is an intrinsic feature in the folding pathway of single-chain Fv fragments

We have studied the equilibrium unfolding and the kinetics of folding and unfolding of an antibody scFv fragment devoid of cis-prolines. An anti-GCN4 scFv fragment carrying a VL lambda domain, obtained by ribosome display, served as the model system together with an engineered destabilized mutant in VH carrying the R66K exchange. Kinetic and equilibrium unfolding experiments indicate that the VH mutation also affects VL unfolding, possibly by partially destabilizing the interface provided by VH, even though the mutation is distant from the interface. Upon folding of the scFv fragment, a kinetic trap is populated whose escape rate is much faster with the more stable VH domain. The formation of the trap can be avoided if refolding is carried out stepwise, with VH folding first. These results show that antibody scFv fragments do not fold by the much faster independent domain folding, but instead form a kinetically trapped off-pathway intermediate, which slows down folding under native conditions. This intermediate is characterized by premature interaction of the unfolded domains, and particularly involving unfolded VH, independent of proline cis-trans isomerization in VL. This work also implies that VH should be a prime target in engineering well behaving antibody fragments.