Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.     

Analysis of the stability of mutant lysozymes at position 15 using X-ray crystallography

His 15 of hen lysozyme is located at the protein surface and is partly buried by the neighboring residues. The side chain of His 15 forms hydrogen bonds with surrounding residues and these hydrogen bonds are somewhat buried. A series of mutant lysozymes at the position 15 (Gly, Ala, Val, and Phe) was prepared, and their stabilities were analyzed by GdnHCl denaturation and X-ray crystallography. The mutants were less stable than the wild type at pH 5.5 and 35 degrees C. In H15G and H15A, X-ray crystallography revealed two fixed water molecules at the mutated region, which formed similar hydrogen bonds to those in the wild type. On the other hand, it was suggested that the hydrogen bonds were disrupted and that several unfavorable van der Waals' contacts occurred in H15V and H15F. Therefore, we concluded that His 15 stabilized the lysozyme structure by forming hydrogen bonds and the best packing with the neighboring residues. Moreover, we found that the method of protein stabilization by increasing the hydrophobicity of an amino acid residue was not always effectively applicable, especially when the residue had formed a hydrogen bond.     

Hydrophobic core substitutions in calbindin D9k: effects on stability and structure

The effects of hydrophobic core mutations on the stability and structure of the four-helix calcium-binding protein, calbindin D9k, have been investigated. Eleven mutations involving eight residues distributed within the hydrophobic core of calbindin D9k were examined. Stabilities were measured by denaturant and thermal induced unfolding monitored by circular dichroism spectroscopy. The mutations were found to exert large effects on the stability with midpoints in the urea induced unfolding varying from 1.8 M for Leu23 --> Gly up to 6.6 M for Val70 --> Leu and free energies of unfolding in the absence of denaturant ranging from 6.6 to 27.4 kJ/mol for the Phe66 --> Ala mutant and the wild-type, respectively. A significant correlation was found between the difference in free energy of unfolding (Delta Delta GNU) and the change in the surface area of the side chain caused by the mutation, in agreement with other studies. Notably, both increases and decreases in side-chain surface area caused quantitatively equivalent effects on the stability. In other words, a correlation between the absolute value of the change in the surface of the side chain and Delta DeltaGNU was observed with a value of approximately 0.14 kJ M-1 A-2. The generality of this observation is discussed. Significant effects on the cooperativity of the unfolding reaction were also observed. However, a correlation between the cooperativity and Delta Delta GNU, which has been reported in other systems as an indication of effects of mutations on the unfolded state, was not observed for calbindin D9k. Despite the large effects on Delta Delta GNU and cooperativity, the structures of the mutants in the native form remained intact as indicated by circular dichroism, NMR, and fluorescence measurements. The structural response to calcium-binding was also conserved. The following paper in this issue [Kragelund, B. B., et al. (1998) Biochemistry 37, 8926-8937] examines the effects of these mutations on the calcium binding properties of calbindin D9k.     

High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Hydrogen bonding stabilizes globular proteins

It is clear that intramolecular hydrogen bonds are essential to the structure and stability of globular proteins. It is not clear, however, whether they make a net favorable contribution to this stability. Experimental and theoretical studies are at odds over this important question. Measurements of the change in conformational stability, delta (delta G), for the mutation of a hydrogen bonded residue to one incapable of hydrogen bonding suggest a stabilization of 1.0 kcal/mol per hydrogen bond. If the delta (delta G) values are corrected for differences in side-chain hydrophobicity and conformational entropy, then the estimated stabilization becomes 2.2 kcal/mol per hydrogen bond. These and other experimental studies discussed here are consistent and compelling: hydrogen bonding stabilizes globular proteins.     

Energetic cost and structural consequences of burying a hydroxyl group within the core of a protein determined from Ala-->Ser and Val-->Thr substitutions in T4 lysozyme

In order to determine the thermodynamic cost of introducing a polar group within the core of a protein, a series of nine Ala-->Ser and 3 Val-->Thr substitutions was constructed in T4 lysozyme. The sites were all within alpha-helices but ranged from fully solvent-exposed to totally buried. The range of destabilization incurred by the Ala-->Ser substitutions was found to be very similar to that for the Val-->Thr replacements. For the solvent-exposed and partly exposed sites the destabilization was modest (approximately less than 0.5 kcal/mol). For the completely buried sites the destabilization was larger, but variable (approximately 1-3 kcal/mol). Crystal structure determinations showed that the Ala-->Ser mutant structures were, in general, very similar to their wild-type counterparts, even though the replacements introduce a hydroxyl group. This is in part because the introduced serines are all within alpha-helices and at congested sites can avoid steric clashes with surrounding atoms by making a hydrogen bond to a backbone carbonyl oxygen in the preceding turn of the helix. The three substituted threonine side chains essentially superimpose on their valine counterparts but display somewhat larger conformational adjustments. The results illustrate how a protein structure will adapt in different ways to avoid the presence of an unsatisfied hydrogen bond donor or acceptor. In the most extreme case, Val 149-->Thr, which is also the most destabilizing variant (delta delta G = 2.8 kcal/mol), a water molecule is incorporated in the mutant structure in order to provide a hydrogen-bonding partner. The results are consistent with the view that many hydrogen bonds within proteins contribute only marginally to stability but that noncharged polar groups that lack a hydrogen-bonding partner are very destabilizing (delta delta G approximately greater than 3 kcal/mol). Supportive of other studies, the alpha-helix propensity of alanine is seen to be higher than that of serine (delta delta G = 0.46 +/- 0.04 kcal/mol), while threonine and valine are similar in alpha-helix propensity.     

Quaternary structure sensitive tyrosine residues in human hemoglobin: UV resonance raman studies of mutants at alpha140, beta35, and beta145 tyrosine

Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA [Nagai, M., et al. (1996) J. Mol. Struct. 379, 65-75; Huang, S., et al. (1997) Biochemistry 36, 6197-6206], but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone. To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here. The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions. The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond. The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible. In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift. In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected. This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes. It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145). These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments.     

Crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34

Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man. We have prepared the mutant containing the replacement Tyr 34 --> Phe (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystallized it in two different crystal forms, orthorhombic and hexagonal. Crystal structures of hMnSOD Y34F have been solved to 1.9 A resolution in a hexagonal crystal form, denoted as Y34Fhex, and to 2.2 A resolution in an orthorhombic crystal form, denoted as Y34Fortho. Both crystal forms give structures that are closely superimposable with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in the wild type and Phe 34 in the mutant very similar in orientation. Therefore, in Y34F, a hydrogen-bonded relay that links the metal-bound hydroxyl to ordered solvent (Mn-OH to Gln 143 to Tyr 34 to H2O to His 30) is broken. Surprisingly, the loss of the Tyr 34 hydrogen bonds resulted in large increases in stability (measured by Tm), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site architecture. The functional role of the side chain hydroxyl of Tyr 34 can be evaluated by comparison of the Y34F mutant with the wild-type hMnSOD. Both wild-type and Y34F had kcat/Km near 10(9) M-1 s-1, close to diffusion-controlled; however, Y34F showed kcat for maximal catalysis smaller by 10-fold than the wild type. In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme. This activity profile and the breaking of the hydrogen-bonding chain at the active site caused by the replacement Tyr 34 --> Phe suggest that Tyr 34 is a proton donor for O2* - reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer. Up to 100 mM buffers in solution failed to enhance catalysis by either Y34F or the wild-type hMnSOD, suggesting that protonation from solution cannot enhance the release of the inhibiting bound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures. The increased thermostability of the mutant Y34F and equal diffusion-controlled activity of Y34F and wild-type enzymes with normal superoxide levels suggest that evolutionary conservation of active-site residues in metalloenzymes reflects constraints from extreme rather than average cellular conditions. This new hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation may apply most strongly to enzyme defenses where the ability to meet extreme conditions directly affects cell survival.     

Active site modifications in a double mutant of liver alcohol dehydrogenase: structural studies of two enzyme-ligand complexes

The oxidation of alcohol to aldehyde by horse liver alcohol dehydrogenase (LADH) requires the transfer of a hydride ion from the alcohol substrate to the cofactor nicotinamide adenine dinucleotide (NAD). A quantum mechanical tunneling contribution to this hydride transfer step has been demonstrated in a number of LADH mutants designed to enhance or diminish this effect [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. The active site double mutant Phe93 --> Trp/Val203 --> Ala shows a 75-fold reduction in catalytic efficiency relative to that of the native enzyme, and reduced tunneling relative to that of either single mutant. We present here two crystal structures of the double mutant: a 2.0 A complex with NAD and the substrate analogue trifluoroethanol and a 2.6 A complex with the isosteric NAD analogue CPAD and ethanol. Changes at the active site observed in both complexes are consistent with reduced activity and tunneling. The NAD-trifluoroethanol complex crystallizes in the closed conformation characteristic of the active enzyme. However, the NAD nicotinamide ring rotates away from the substrate, toward the space vacated by replacement of Val203 with the smaller alanine. Replacement of Phe93 with the larger tryptophan also produces unfavorable steric contacts with the nicotinamide carboxamide group, potentially destabilizing hydrogen bonds required to maintain the closed conformation. These contacts are relieved in the second complex by rotation of the CPAD pyridine ring into an unusual syn orientation. The resulting loss of the carboxamide hydrogen bonds produces an open conformation characteristic of the apoenzyme.     

Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease virus infection in chickens

Presteady and steady-state kinetic results on the interactions of a wild-type, and the mutant glucoamylases Trp52-->Phe and Trp317-->Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trp120-->Phe and Glu180-->Gln, and with results obtained from structure energy minimization calculations based on known three-dimensional structural data. All results are in accordance with a three-step reaction model involving two steps in the substrate binding and a rate-determining catalytic step. Trp317 and Glu180 belong to different subsites, but are placed on the same flank of the active site (beta-flank). The Trp317-->Phe and the Glu180-->Gln mutants show almost identical kinetic results: weakening of the substrate binding, mainly caused by changes in the second reaction step, and practically no change of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the substrate occurs in the Michaelis complexes of each of these mutants. These results indicate that important interactions of the active site may be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the substrate binding mode of maltose and of longer substrates. Trp52 and Trp120 each interact with the catalytic acid, Glu179, and are placed on the flank (alpha-flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52-->Phe and Trp120-->Phe mutants show kinetic results similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All together, the substrate binding mechanism seems to involve an initial enzyme-substrate complex, in which the beta-flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg305 and Glu180 of the beta-flank are established and the correct alignment on the alpha-flank of Glu179, the general acid catalyst, governed by its flexible interactions with Trp52 and Trp120, occurs.     

Crystal structure and enzyme mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni

Bacterial Delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been intensively studied as a prototype for understanding an enzyme-catalyzed allylic rearrangement involving intramolecular proton transfer. Asp38 serves as a general base to abstract the proton from the steroid C4-H, which is a much stronger base than the carboxyl group of this residue. This unfavorable proton transfer requires 11 kcal/mol of energy which has to be provided by favorable interactions between catalytic residues and substrate in the course of the catalytic reaction. How this energy is provided at the active site of KSI has been a controversial issue, and inevitably the enzyme mechanism is not settled. To resolve these issues, we have determined the crystal structure of this enzyme at 2.3 A resolution. The crystal structure revealed that the active site environment of P. testosteroni KSI is nearly identical to that of Pseudomonas putida KSI, whose structure in complex with a reaction intermediate analogue we have determined recently. Comparison of the two structures clearly indicates that the two KSIs should share the same enzyme mechanism involving the stabilization of the dienolate intermediate by the two direct hydrogen bonds to the dienolate oxyanion, one from Tyr14 OH and the other from Asp99 COOH. Mutational analysis of the two residues and other biochemical data strongly suggest that the hydrogen bond of Tyr14 provides the more significant contribution than that of Asp99 to the requisite 11 kcal/mol of energy for the catalytic power of KSI.     

Effects of hydrogen bonding to a bacteriochlorophyll-bacteriopheophytin dimer in reaction centers from Rhodobacter sphaeroides

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner.     

Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides

Site-directed mutagenesis has been used to examine the function of a highly conserved aromatic residue, alpha Trp43, in the light-harvesting 1 antenna of the photosynthetic bacterium Rhodobacter sphaeroides. In this antenna alpha Trp43 is thought to be located near the putative binding site for bacteriochlorophyll; in this work it was changed to both Tyr and Phe, and in each case the main near-infrared absorbance peak was shifted to the blue, from 876 nm to 865 nm and then to 853 nm, respectively. Resonance Raman spectroscopy of the resulting complexes shows a shift of one component of the 1640-cm-1 peak to 1632 cm-1 for the Tyr mutant and to 1660 cm-1 for the Phe mutant. This demonstrates a strengthening of an existing H bond for the Tyr change and a breakage of this bond for the change to Phe. The 1640-cm-1 peak has been previously assigned to H-bonded C2 acetyl carbonyl groups of both bacteriochlorophylls in the light-harvesting 1 antenna dimer [Robert, B. & Lutz, M. (1985) Biochim. Biophys. Acta 807, 10-21]. These results indicate that one of these H bonds is to alpha Trp43, placing this residue in close proximity to the bacteriochlorophyll a macrocycle with which it interacts. The existence of this bond places constraints on the conformation of the alpha polypeptide, and a model of an alpha beta heterodimer is presented incorporating these data.     

Introduction of a proline residue into position 31 of the loop of the dimeric 4-alpha-helical protein ROP causes a drastic destabilization

The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993). The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.     

Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis

Antibodies directed to the A2 domain of factor VIII (fVIII) are usually an important component of the polyclonal response in patients who have clinically significant inhibitory antibodies to fVIII. A major determinant of the A2 epitope has been located by homolog scanning mutagenesis using recombinant hybrid human/porcine fVIII molecules to a sequence bounded by Arg484-Ile508 (Healey, J. F. , Lubin, I. M., Nakai, H., Saenko, E. L., Hoyer, L. W., Scandella, D. , and Lollar, P. (1995) J. Biol. Chem. 270, 14505-14509). Within this region, human residues Arg484, Pro485, Tyr487, Ser488, Arg489, Pro492, Val495, Phe501, and Ile508 differ from porcine fVIII. We stably expressed in mammalian cells nine active B-domainless human fVIII molecules containing single alanine substitutions at these sites. Their inhibition by a murine anti-A2 monoclonal antibody, monoclonal antibody (mAb) 413, and by three A2-specific alloimmune and two A2-specific autoimmune human inhibitor plasmas was measured by the Bethesda assay. The inhibition of Arg484 --> Ala, Tyr487 --> Ala, Arg489 --> Ala, and Arg492 --> Ala by mAb413 was reduced by greater than 90% compared with wild-type, B-domainless human fVIII. mAb413 inhibited the most severely affected mutant, Arg489 --> Ala, 0.01% as well as wild-type fVIII. For all five patient plasmas, the Tyr487 --> Ala mutant displayed the greatest reduction in inhibition. The inhibition of the Tyr487 --> Ala mutant by these antibodies ranged from 10% to 20% that of wild-type fVIII. The inhibition of the Ser488 --> Ala, Arg489 --> Ala, Pro492 --> Ala, Val495 --> Ala, Phe501 --> Ala, and Ile508 --> Ala mutants by most of the plasmas also was significantly reduced. In contrast, the Arg484 --> Ala and Pro485 --> Ala mutants were relatively unaffected. Thus, although mAb413 binds to the same region as human A2 inhibitors, it recognizes a different set of amino acid side chains. The side chains recognized by human A2 inhibitors appear to be similar, despite the differing immune settings that give rise to fVIII alloantibodies and autoantibodies.     

Helix-capping interaction in lambda Cro protein: a free energy simulation analysis

The stability mutant Tyr-26-->Asp was studied in the Cro protein from bacteriophage lambda using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 +/- 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interaction with the amino terminus of the third alpha-helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix-capping interaction and to examine the results in light of previous explanations of stabilizing helix caps--namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macrodipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge-dipole interactions and may be employed to advantage for molecular design.     

Tyrosine quenching of tryptophan phosphorescence in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Tyrosine is known to quench the phosphorescence of free tryptophan derivatives in solution, but the interaction between tryptophan residues in proteins and neighboring tyrosine side chains has not yet been demonstrated. This report examines the potential role of Y283 in quenching the phosphorescence emission of W310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by comparing the phosphorescence characteristics of the wild-type enzyme to that of appositely designed mutants in which either the second tryptophan residue, W84, is replaced with phenylalanine or Y283 is replaced by valine. Phosphorescence spectra and lifetimes in polyol/buffer low-temperature glasses demonstrate that W310, in both wild-type and W84F (Trp84-->Phe) mutant proteins, is already quenched in viscous low-temperature solutions, before the onset of major structural fluctuations in the macromolecule, an anomalous quenching that is abolished with the mutation Y283V (Tyr283-->Val). In buffer at ambient temperature, the effect of replacing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 micros to 2.5 ms, a 50-fold enhancement that again emphasizes how W310 emission is dominated by the local interaction with Y283. Tyr quenching of W310 exhibits a strong temperature dependence, with a rate constant kq = 0.1 s(-1) at 140 K and 2 x 10(4) s(-1) at 293 K. Comparison between thermal quenching profiles of the W84F mutant in solution and in the dry state, where protein flexibility is drastically reduced, shows that the activation energy of the quenching reaction is rather small, Ea < or = 0.17 kcal mol(-1), and that, on the contrary, structural fluctuations play an important role on the effectiveness of Tyr quenching. Various putative quenching mechanisms are examined, and the conclusion, based on the present results as well as on the phosphorescence characteristics of other protein systems, is that Tyr quenching occurs through the formation of an excited-state triplet exciplex.     

Stabilization of ribonuclease HI from Thermus thermophilus HB8 by the spontaneous formation of an intramolecular disulfide bond

To identify factors that contribute to the thermal stability of ribonuclease HI (RNase HI) from Thermus thermophilus HB8, protein variants with a series of carboxyl-terminal truncations and Cys --> Ala mutations were constructed, and their thermal denaturations were analyzed by CD. The results indicate that Cys41 and Cys149 contribute to the protein stability, probably through the formation of a disulfide bond. Peptide mapping analysis for the mutant protein with only two cysteine residues, at positions 41 and 149, indicated that this disulfide bond is partially formed in a protein purified from Escherichia coli in the absence of a reducing reagent but is fully formed in a thermally denatured protein. These results suggest that the thermal stability of T. thermophilus RNase HI, determined in the absence of a reducing reagent, reflects that of an oxidized form of the protein. Comparison of the thermal stabilities and the enzymatic activities of the wild-type and truncated proteins, determined in the presence and absence of a reducing reagent, indicates that the formation of this disulfide bond increases the thermal stability of the protein by 6-7 degreesC in Tm and approximately 3 kcal/mol in DeltaG without seriously affecting the enzymatic activity. Since T. thermophilus RNase HI is present in a reducing environment in cells, this disulfide bond probably is not formed in vivo but is spontaneously formed in vitro in the absence of a reducing reagent.     

S100A1 utilizes different mechanisms for interacting with calcium-dependent and calcium-independent target proteins

While previous studies have identified target proteins that interact with S100A1 in a calcium-dependent manner as well as target proteins that interact in a calcium-independent manner, the molecular mechanisms of S100A1-target protein interaction have not been elucidated. In this study, point and deletion mutants of S100A1 were used to investigate the contribution of carboxyl terminal amino acids to S100A1 interaction with calcium-dependent and calcium-independent target proteins. First, a recombinant rat S100A1 protein (recS100A1) expressed in bacteria exhibited physical and chemical properties indistinguishable from native S100A1. Next, proteins lacking the carboxyl-terminal nine residues of recS100A1 (Delta85-93), or containing alanine substitutions at Phe 88 (F88A), Phe 89 (F89A), or Trp 90 (W90A), both Phe 88 and Phe 89 (F88/89A), or all three aromatic residues (F88/89A-W90A) were recombinantly expressed. Like recS100A1, F88A, F89A, and W90A proteins interacted with phenyl-Sepharose in a calcium-dependent manner. However, the Delta85-93 protein did not interact with phenyl-Sepharose, indicating that a phenyl-Sepharose-binding region (PSBR) of recS100A1 had been disrupted. The F88/89A and F88/89A-W90A proteins exhibited reduced calcium-dependent interaction with phenyl-Sepharose when compared with recS100A1, demonstrating that the carboxyl-terminal aromatic residues Phe 88, Phe 89, and Trp 90 comprise the PSBR of S100A1. Fluorescence studies showed that the Delta85-93 protein exhibited reduced calcium-dependent interaction with the dodecyl CapZ peptide, TRTK, while W90A bound TRTK with a Kd of 5.55 microM. These results demonstrate that the calcium-dependent target protein-binding site and the PSBR are indistinguishable. In contrast to the calcium-dependent target TRTK, activation of the calcium-independent target protein aldolase A by the point and deletion mutant S100A1s was indistinguishable from native S100A1. These results demonstrate that carboxyl-terminal residues are not required for S100A1 modulation of calcium-independent target protein aldolase A. Alltogether, these results indicate that S100A1 utilizes distinct mechanisms for interaction with calcium-independent and calcium-dependent target proteins.     

Metabolic control during preimplantation mammalian development

The stability properties of six natural mutants of the TEM-1 beta-lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the N delta of His-164 and the O delta of Asp-179. The properties of the G238S + E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon.