Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.     

Folding of a three-stranded coiled coil

Coiled coils consist of two or more amphipathic a-helices wrapped around each other to form a superhelical structure stabilized at the interhelical interface by hydrophobic residues spaced in a repeating 3-4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which association and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three-stranded coiled coil. The designed 29-residue peptide LZ16A was shown previously to be in a concentration-dependent equilibrium between unfolded monomer (M), folded dimer (D), and folded trimer (T). We show by time-resolved fluorescence change experiments that folding of LZ16A to D and T can be described by 2M (k1)<==>(k(-1)) D and M + D (k2)<==>(k(-2)) T. The following rate constants were determined (25 degrees C, pH 7): k1 = 7.8 x 10(4) M(-1) s(-1), k(-1) = 0.015 s(-1), k2 = 6.5 x 10(5) M(-1) s(-1), and k(-2) = 1.1 s(-1). In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far-ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to K(D) = k1/2k(-1) and K(T) = k2/k(-2). Furthermore, pulsed hydrogen-exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are compatible with a two-step reaction in which a subpopulation of association competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.     

Muscle fatty acid-binding protein

Muscle or heart fatty acid-binding protein is a low molecular weight protein that binds long-chain fatty acids in the cytosol of muscle tissues. The three-dimensional structure of the human, bovine and insect proteins are known, either via X-ray or NMR techniques. The folding of the protein closely resembles that of the other FABPs: ten anti-parallel beta-strands are arranged to form a clam shell, closed at one end by two alpha-helices. This arrangement allows the formation of an internal cavity where the fatty acid can be accommodated, protected and isolated from the external environment. The fatty acid in the protein interior is stabilized by electrostatic and hydrogen bond interactions of its carboxylic head with charged or polar residues of the protein and by interactions of its tail with hydrophobic residues. The three-dimensional structure of different fatty acid-protein complexes along with molecular dynamics simulations are now providing insight into the molecular details of the specificity of the ligand binding.     

Hydrogen-deuterium exchange in free and prodomain-complexed subtilisin

Residue-specific exchange rates of 223 amide protons in free and prodomain-complexed subtilisin were determined in order to understand how the prodomain binding affects the energetics of subtilisin folding. In free subtilisin, amide protons can be categorized according to exchange rate: 74 fast exchangers (rates > or = 1 h(-1)); 52 medium exchangers (rates between 1 h(-1) and 1 day(-1)); 31 slow exchangers (rates between 1 day(-1) and 0.001 day(-1)). The remaining 66 amide proteins did not exchange detectibly over 9 months (k(obs) < year(-1)) and were denoted as core protons. Core residues occur throughout the main structural elements of subtilisin. Prodomain binding results in high protection factors (100-1000) in the central beta-sheet, particularly in the vicinity of beta-strands S5, S6, and S7 and the connecting loops between them. These connecting loops provide the ligands to the cation at metal site B. Overall, prodomain binding seems to facilitate the organization of the entire central beta-sheet and alpha-helix C in the left-handed crossover connection between beta-strands two and three. It also appears to facilitate the isomerization of multiple prolines late in folding, allowing the formation of metal site B. The gain of stability region around site B comes at the cost of stability in regions more distal to prodomain binding: the C-terminal alpha-helix H and the N-terminal alpha-helices A and B. The acceleration of exchange in these regions by prodomain binding reveals an antagonism between the folding intermediate and the full native structure. This antagonism helps to explain why the prodomain is needed to stabilize the folding intermediate as well as why the unfolding of free subtilisin seldom occurs via this intermediate.     

Analysis of folding and unfolding reactions of cytochrome b5

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.     

The slow folding reaction of barstar: the core tryptophan region attains tight packing before substantial secondary and tertiary structure formation and final compaction of the polypeptide chain

The slow folding of a single tryptophan-containing mutant of barstar has been studied in the presence of 2 M urea at 10 degrees C, using steady state and time-resolved fluorescence methods and far and near-UV CD measurements. The protein folds in two major phases: a fast phase, which is lost in the dead time of measurement during which the polypeptide collapses to a compact form, is followed by a slow observable phase. During the fast phase, the rotational correlation time of Trp53 increases from 2.2 ns to 7.2 ns, and its mean fluorescence lifetime increases from 2.3 ns to 3.4 ns. The fractional changes in steady-state fluorescence, far-UV CD, and near-UV CD signals, which are associated with the fast phase are, respectively, 36 %, 46 %, and 16 %. The product of the fast phase can bind the hydrophobic dye ANS. These observations together suggest that the folding intermediate accumulated at the end of the fast phase has: (a) about 20 % of the native-state secondary structure, (b) marginally formed or disordered tertiary structure, (c) a water-intruded and mobile protein interior; and (d) solvent-accessible patches of hydrophobic groups. Measurements of the anisotropy decay of Trp53 suggest that it undergoes two types of rotational motion in the intermediate: (i) fast (tau(r) approximately 1 ns) local motion of its indole side-chain, and (ii) a slower (tau(r) approximately 7.2 ns) motion corresponding to global tumbling of the entire protein molecule. The ability of the Trp53 side-chain to undergo fast local motion in the intermediate, but not in the fully folded protein where it is completely buried in the hydrophobic core, suggests that the core of the intermediate is still poorly packed. The global tumbling time of the fully folded protein is faster at 5.6 ns, suggesting that the volume of the intermediate is 25 % more than that of the fully folded protein. The rate of folding of this intermediate to the native state, measured by steady-state fluorescence, far-UV CD, and near-UV CD, is 0.07(+/-0.01) min(-1) This rate compares to a rate of folding of 0.03(+/-0.005) min(-1), determined by double-jump experiments which monitor directly formation of native protein; and to a rate of folding of 0.05 min(-1), when determined from time-resolved anisotropy measurements of the long rotational correlation time, which relaxes from an initial value of 7.2 ns to a final value of 5. 6 ns as the protein folds. On the other hand, the amplitude of the short correlation time decreases rapidly with a rate of 0.24(+/-0.06) min(-1). These results suggest that tight packing of residues in the hydrophobic core occurs relatively early during the observable slow folding reaction, before substantial secondary and tertiary structure formation and before final compaction of the protein.     

Trapping the on-pathway folding intermediate of Im7 at equilibrium

The four-helical protein Im7 folds via a rapidly formed on-pathway intermediate (k(UI)=3000 s(-1) at pH 7.0, 10 degrees C) that contains three (helices I, II and IV) of the four native alpha-helices. The relatively slow (k(IN)=300 s(-1)) conversion of this intermediate into the native structure is driven by the folding and docking of the six residue helix III onto the developing hydrophobic core. Here, we describe the structural properties of four Im7* variants designed to trap the protein in the intermediate state by disrupting the stabilising interactions formed between helix III and the rest of the protein structure. In two of these variants (I54A and L53AI54A), hydrophobic residues within helix III have been mutated to alanine, whilst in the other two mutants the sequence encompassing the native helix III was replaced by a glycine linker, three (H3G3) or six (H3G6) residues in length. All four variants were shown to be monomeric, as judged by analytical ultracentrifugation, and highly helical as measured by far-UV CD. In addition, all the variants denature co-operatively and have a stability (DeltaG(UF)) and buried hydrophobic surface area (M(UF)) similar to those of the on-pathway kinetic intermediate. Structural characterisation of these variants using 1-anilino-8-napthalene sulphonic acid (ANS) binding, near-UV CD and 1D (1)H NMR demonstrate further that the trapped intermediate ensemble is highly structured with little exposed hydrophobic surface area. Interestingly, however, the structural properties of the variants I54A and L53AI54A differ in detail from those of H3G3 and H3G6. In particular, the single tryptophan residue, located near the end of helix IV, and distant from helix III, is in a distinct environment in the two sets of mutants as judged by fluorescence, near-UV CD and the sensitivity of tryptophan fluorescence to iodide quenching. Overall, the results confirm previous kinetic analysis that demonstrated the hierarchical folding of Im7 via an on-pathway intermediate, and show that this species is a highly helical ensemble with a well-formed hydrophobic core. By contrast with the native state, however, the intermediate ensemble is flexible enough to change in response to mutation, its structural properties being tailored by residues in the sequence encompassing the native helix III.     

Tight binding of inhibitors to bovine bc1 complex is independent of the Rieske protein redox state. Consequences for semiquinone stabilization in the quinol oxidation site

To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.     

Tuning lambda6-85 towards downhill folding at its melting temperature

The five-helix bundle lambda6-85* is a fast two-state folder. Several stabilized mutants have been reported to fold kinetically near-downhill or downhill. These mutants undergo a transition to two-state folding kinetics when heated. It has been suggested that this transition is caused by increased hydrophobicity at higher temperature. Here we investigate two histidine-containing mutants of lambda6-85* to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable lambdaHA is the fastest-folding lambda repressor to date (k(f)(-1) approximately k(obs)(-1)=2.3 micros at 44 degrees C). It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. lambdaHG has a weakened hydrophobic core. It is less stable than some slower folding mutants of lambda6-85*, and it has more exposed hydrophobic surface area in the folded state. This mutant nonetheless folds very rapidly, and has the non-exponential folding kinetics of an incipient downhill folder even at the unfolding midpoint (k(m)(-1) approximately 2 micros, k(a)(-1)=15 micros at 56 degrees C). We also compare the thermodynamic melting transition of lambdaHG with the nominal two-state folding mutant lambdaQG, which has a similar melting temperature. Unlike lambdaQG, lambdaHG yields fluorescence wavelength-dependent cooperativities and probe-dependent melting temperatures. This result combined with previous work shows that the energy landscapes of lambda repressor mutants support all standard folding mechanisms.     

Heat capacity effects in protein folding and ligand binding: a re-evaluation of the role of water in biomolecular thermodynamics

Large "anomalous" heat capacity (DeltaC(p)) effects are a common feature of the thermodynamics of biomolecular interactions in aqueous solution and, as a result of the improved facility for direct calorimetric measurements, there is a growing body of experimental data for such effects in protein folding, protein-protein and protein-ligand interactions. Conventionally such heat capacity effects have been ascribed to hydrophobic interactions, and there are some remarkably convincing demonstrations of the usefulness of this concept. Nonetheless, there is also increasing evidence that hydrophobic interactions are not the only possible source of such effects. Here we re-evaluate the possible contributions of other interactions to the heat capacity changes to be expected for cooperative biomolecular folding and binding processes, with particular reference to the role of hydrogen bonding and solvent water interactions. Simple models based on the hydrogen-bonding propensity of water as a function of temperature give quantitative estimates of DeltaC(p) that compare well with experimental observations for both protein folding and ligand binding. The thermodynamic contribution of bound waters in protein complexes is also estimated. The prediction from simple lattice models is that trapping of water in a complex should give more exothermic binding (DeltaDeltaH-6 to -12 kJ mol(-1)) with lower entropy (DeltaDeltaS(0) approximately -11 J mol(-1) K(-1)) and more negative DeltaC(p) (by about -75 J mol(-1) K(-1)) per water molecule. More generally, it is clear that significant DeltaC(p) effects are to be expected for any macromolecular process involving a multiplicity of cooperative weak interactions of whatever kind.     

Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange

The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding.     

Quantitative evaluation of each catalytic subsite of cathepsin B for inhibitory activity based on inhibitory activity-binding mode relationship of epoxysuccinyl inhibitors by X-ray crystal structure analyses of complexes

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.     

Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.     

A residual structure in unfolded intestinal fatty acid binding protein consists of amino acids that are neighbors in the native state

Much of the recent effort in protein folding has focused on the possibility that residual structures in the unfolded state may provide an initiating site for protein folding. This hypothesis is difficult to test because of the weak stability and dynamic behavior of these structures. This problem has been simplified for intestinal fatty acid binding protein (IFABP) by incorporating fluorinated aromatic amino acids during synthesis in Escherichia coli. Only the labeled residues give signals by (19)F NMR, and the 1D spectra can be assigned in both the native and unfolded states by site-directed mutagenesis. One of the two tryptophans (W82), one of the four tyrosines (Y70), and at least four of the eight phenylalanines (including F68 and F93) of IFABP are involved in a structure that is significantly populated at concentrations of urea that unfold the native structure by fluorescence and CD criteria. These residues are nonlocal in sequence and also contact each other in the native structure. Thus, a template of nativelike hydrophobic contacts in the unfolded state may serve as an initiating site for folding this beta-sheet protein.     

Crystal structure of the cytokine interleukin-1 beta.

The crystal structure of human recombinant interleukin-1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R-factor of 42.3%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557-563, for soybean trypsin inhibitor.     

Kinetics of α-globin binding to α-hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of hemichrome folding intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ～100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.     

Swapping core residues in homologous proteins swaps folding mechanism

Rat intestinal fatty acid binding protein (IFABP) displays an intermediate with little if any secondary structure during unfolding, while the structurally homologous rat ileal lipid binding protein (ILBP) displays an intermediate during unfolding with nativelike secondary structure. Double-jump experiments indicate that these intermediates are on the folding path for each protein. To test the hypothesis that differences in the number of buried hydrophobic atoms in a folding initiating site are responsible for the different types of intermediates observed for these proteins, two mutations (F68C-IFABP and C69F-ILBP) were made that swapped a more hydrophobic residue for a more hydrophilic residue in the respective cores of these two proteins. F68C-IFABP followed an unfolding path identical to that of WT-ILBP with an intermediate that showed nativelike secondary structure, whereas C69F-ILBP followed an unfolding path that was identical to that of WT-IFABP with an intermediate that lacked secondary structure. Further, a hydrophilic residue was introduced at an identical hydrophobic structural position in both proteins (F93S-IFABP and F94S-ILBP). Replacement of phenylalanine with serine at this site led to the appearance of an intermediate during refolding that lacked secondary structure for both proteins that was not detected for either parental protein. Altering the chemical characteristics and/or size of residues within an initiating core of hydrophobic interactions is critical to the types of intermediates that are observed during the folding of these proteins.     

Rapid-mix and chemical quench studies of ferredoxin-reduced stearoyl-acyl carrier protein desaturase

Stearoyl-ACP Delta9 desaturase (Delta9D) catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (approximately 95% per Delta9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme-substrate complex with O(2) and the oxidized enzyme-substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme-substrate complex, an exponential burst phase (k(burst) = 95 s(-1)) of product formation accounted for approximately 90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (k(linear) = 4.0 s(-1)) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme-substrate complex with excess reduced Fd, a slower, linear rate (k(obsd) = 3.4 s(-1)) of product formation was observed over approximately 1.5 turnovers per Delta9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on k(burst) ((D)k(burst) approximately 1.5) and k(linear) (D)k(linear) approximately 1.4), indicating C-H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Delta9D catalysis.     

Galactosyl transfer catalyzed by thermostable beta-glycosidases from Sulfolobus solfataricus and Pyrococcus furiosus: kinetic studies of the reactions of galactosylated enzyme intermediates with a range of nucleophiles

The transfer of a galactosyl group from an enzyme to a number of neutral primary alcohols, phenol and azide has been studied during the reactions at 80 degrees C of thermostable beta-glycosidases from Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) with 2-nitrophenyl beta-D-galactopyranoside or lactose (4-O-beta-D-galactopyranosyl D-glucopyranose) as substrates. The rate constant ratios, k(Nu)/k(water), for partitioning of the galactosylated enzyme intermediates between reaction with nucleophiles (k(Nu), M(-1) s(-1)) and water (k(water), s(-1)) have been determined from the difference in the initial velocities of the formation of 2-nitrophenol or D-glucose, and D-galactose. The results show that hydrophobic bonding interactions contribute approximately 8 kJ mol(-1) to the stabilization of the transition state for the reaction of galactosylated enzyme intermediates of Ss beta Gly and CelB with 1-butanol, compared to the transition state for the enzymatic reaction with methanol. The leaving group/nucleophile binding sites of Ss beta Gly and CelB appear about 0.8 times as hydrophobic as n-octanol. Values of k(Nu)/k(water) for reactions of galactosylated Ss beta Gly with ethanol and substituted derivatives of ethanol show no clear dependence on the pK(a) of the primary hydroxy group of these nucleophiles in the pK(a) range 12.4-16.0. The binding of phenol with the galactosylated enzyme intermediates of Ss beta Gly and CelB occurs in a form that is mainly nonproductive pertaining to beta-galactoside synthesis. Neither enzyme catalyzes galactosyl transfer to azide ion. A model is proposed for the interaction of neutral nucleophiles at an extended acceptor site of the galactosylated enzymes.     

Distinct kinetics for binding of the CD46 and SLAM receptors to overlapping sites in the measles virus hemagglutinin protein

Measles virus (MV) is a human pathogen using two distinct cell surface receptors for entry into host cells. We present here a comparative analysis for binding of the MV receptors CD46 and SLAM to the measles virus hemagglutinin protein (MVH, Edmonston strain). Soluble monomeric and dimeric MVH variants were prepared in mammalian cells and their conformation assessed using a panel of monoclonal antibodies. The two receptor molecules specifically bound to the MVH protein with distinct binding modes. The association rate (k(a)) for SLAM binding to MVH was very low ( approximately 3000 m(-1)s(-1)), about 20 times lower that the k(a) determined for CD46 binding. However, SLAM bound tighter to the virus protein than CD46, as revealed by a 5-fold lower dissociation rate (k(d), approximately 1.5 x 10(-3) s(-1)). These data suggest that the SLAM receptor binds to a less accessible and more hydrophobic surface on MVH than the CD46 receptor, as illustrated in a binding model. Despite the differences in kinetics, receptor competition binding experiments revealed that they recognize overlapping sites in MVH. Indeed, a panel of anti-MVH monoclonal antibodies equally inhibited binding of both receptor molecules. The similar immune reactivity of the two receptor binding sites suggests that the shift in receptor usage by MV may not be driven by immune responses.