Single Tryptophan Mutants Research Articles

Effects of Single-Tryptophan Mutations on R67 Dihydrofolate Reductase

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers clinical resistance to the antibacterial drug trimethoprim. This enzyme shows no sequence or structural homology to the chromosomal DHFRs. The active form of the protein is a homotetramer possessing D(2) symmetry and a single active-site pore. Two tryptophans occur per monomer: W38 and its symmetry-related residues (W138, W238, and W338) occur at the dimer-dimer interfaces, while W45 and its symmetry-related partners (W145, W245, and W345) occur at the monomer-monomer interfaces. Two single-tryptophan mutant genes were constructed to determine the structural and functional consequences of four mutations per tetramer. The W45F mutant retains full enzyme activity and the fluorescence environment of the unmutated W38 residues clearly monitors ligand binding and a pH dependent tetramer right harpoon over left harpoon 2 dimers equilibrium. In contrast, four simultaneous W38F mutations at the dimer-dimer interfaces result in tetramer destabilization. The ensuing dimer is relatively inactive, as is dimeric wild-type R67 DHFR. A comparison of emission spectra indicates the fluorescent signal of wild-type R67 DHFR is dominated by the contribution from W38. Equilibrium unfolding/folding curves at pH 5.0, where all protein variants are dimeric, indicate the environment monitored by the W38 residue is slightly less stable than the environment monitored by the W45 residue.

Temperature-dependent Chaperone Activity and Structural Properties of Human αA- and αB-crystallins

The chaperone activity and biophysical properties of recombinant human alphaA- and alphaB-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of alphaA-crystallin markedly improves with an increase in temperature, the activity of alphaB homopolymer appears to change very little upon heating. Compared with alphaB-crystallin, the alphaA-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the alphaB homopolymer appears to be modestly (two times or less) more potent chaperone than alphaA homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, alphaA- and alphaB-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, alphaA-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for alphaB-crystallin. The correlation between temperature-dependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of alpha-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of alpha-crystallin.

Conformational changes induced in the Tet repressor protein TetR(B) upon operator or anhydrotetracycline binding as revealed by time-resolved fluorescence spectroscopy on single tryptophan mutants.

We have analyzed the tryptophan (trp) fluorescence-decay kinetics of single trp mutants of the Tet repressor protein in the free, the tet operator and anhydrotetracycline (atc)-bound states. The position of the single trp varies between residues 164 and 171, in close proximity to one entrance of the tetracycline-binding pocket. A good fit of the trp fluorescence decay needed generally three exponentials. The decay times vary with detection wavelength, the extent of this variation being correlated to the variation of the emission maximum. Quenching experiments with neutral (acrylamide), cationic (N-methylpyridinium chloride) and anionic quencher (KI) support the interpretation of the three fluorescence components within a conformer model. Operator and atc binding change the ratio of the relative amplitudes of the medium- and long-lived component, thus pointing to structural changes as indicated also by the changes in decay time. Since the fluorescence decay is different between the free, atc- and operator-bound states we conclude that the protein structure is different in each of these three states. The fluorescence quenching constants reflect not only the variation in solvent exposure with position, but also the fact that the net surface charge in this region is negative, because the quenching constants by the cationic quencher are up to 10-fold higher. The atc fluorescence appears to decay monoexponentially with about the same decay time for all mutants, except W170, in which the trp residue sterically interferes with atc.

Refolded Outer Membrane Protein A of Escherichia coliForms Ion Channels with Two Conductance States in Planar Lipid Bilayers

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood. Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial. We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements. In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels. These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane. The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels. The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA. Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers.

Evaluation of Lipid Exposure of Tryptophan Residues in Membrane Peptides and Proteins

Fluorescence quenching is used to gain information on the exposure of tryptophan residues to lipid in membrane-bound proteins and peptides. A protocol is developed to calculate this exposure, based on a comparison of quenching efficiency and of a fluorescence lifetime (or quantum yield) measured for a protein and for a model tryptophan-containing compound. Various methods of analysis of depth-dependent quenching are compared and three universal measures of quenching profile are derived. One of the measures, related to the area under profile, is used to estimate quenching efficiency. The method is applied to single tryptophan mutants of a membrane-anchoring nonpolar peptide of cytochrome b5 and of an outer membrane protein A. Analysis of quenching of the cytochrome's nonpolar peptide by a set of four brominated lipids reveals a temperature-controlled reversible conformational change, resulting in increased exposure of tryptophan to lipid and delocalization of its transverse position. Kinetic quenching profiles and fluorescence binding kinetics reported by Kleinschmidt et al. (Biochemistry (1999) 38, 5006–5016) were analyzed to extract information on the relative exposure of tryptophan residues during folding of an outer membrane protein A. Trp-102, which translocates across the bilayer, was found to be noticeably shielded from the lipid environment throughout the folding event compared to Trp-7, which remains on the cis side. The approach described here provides a new tool for studies of low-resolution structure and conformational transitions in membrane proteins and peptides.

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.

Unfolding and Refolding of Human Glyoxalase II and its Single-tryptophan Mutants

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a “zinc binding motif” (T/SHXHX 57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent β-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved “zinc binding motif” is located in a flexible region of the active site in which Zn(II) participates in catalysis.

Adventures in Membrane Protein Topology: A STUDY OF THE MEMBRANE-BOUND STATE OF COLICIN E1

The molecular aggregate size of the closed state of the colicin E1 channel was determined by fluorescence resonance energy transfer experiments involving a fluorescence donor (three tryptophans, wild-type protein) and a fluorescence acceptor (5-(((acetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (AEDANS), Trp-deficient protein). There was no evidence of energy transfer between the donor and acceptor species when bound to membrane large unilamellar vesicles. These experiments led to the conclusion that the colicin E1 channel is monomeric in the membrane-bound closed channel state. Experiments were also conducted to study the membrane topology of the closed colicin channel in membrane large unilamellar vesicles using acrylamide as the membrane-impermeant, nonionic quencher of tryptophan fluorescence in a battery of single tryptophan mutant proteins. Furthermore, additional fluorescence parameters, including fluorescence emission maximum, fluorescence quantum yield, and fluorescence decay times, were used to assist in mapping the topology of the closed channel. Results suggest that the closed channel comprises most of the polypeptide of the channel domain and that the hydrophobic anchor domain does not transverse the membrane bilayer but nonetheless is deeply embedded within the hydrocarbon core of the membrane. Finally, a model is proposed which features at least two states that are in rapid equilibrium with each other and in which one state is more heavily populated than the other.

Binding of Zn(II), Cu(II), and Fe(II) ions to alzheimer's Aß peptide studied by fluorescence

Binding of Zn(II), Cu(II) and Fe(II) ions to Aβ1-40, Aβ1-42 and a single tryptophan mutant of Aβ1-40 in solution at pH 7.4 was studied by fluorescent titration. Job plots and fitting of titration curves revealed formation of 1:1 and 1:2 peptide-metal complexes. For dimeric peptides Aβ1-40 and AβF4W the order of metal to peptide affinities is Fe < Cu > Zn, which is in agreement with the Irving-Williams series of complex stability. The affinity of Aβ1-42 for Fe increases dramatically upon aggregation: K d changes from ca. 100 to ca. 0.2 μM.

Membrane protein-ligand interactions in Escherichia coli vesicles and living cells monitored via a biosynthetically incorporated tryptophan analogue.

This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells. This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. coli (EII(mtl)), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII(mtl). By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII(mtl) was achieved. The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII(mtl) containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment [Schlesinger, R. (1968) J. Biol. Chem. 243, 3877-3883]. Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction. Of the five single-tryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding. Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%). The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions.

A Fluorescence Investigation of the Active Site ofPseudomonas aeruginosa Exotoxin A

Single tryptophan mutant proteins of a catalytically active domain III recombinant protein (PE24) from Pseudomonas aeruginosa exotoxin A were prepared by site-directed mutagenesis. The binding of the dinucleotide substrate, NAD+, to the PE24 active site was studied by exploiting intrinsic tryptophan fluorescence for the wild-type, single Trp, and tryptophan-deficient mutant proteins. Various approaches were used to study the substrate binding process, including dynamic quenching, CD spectroscopy, steady-state fluorescence emission analysis, NAD+-glycohydrolase activity, NAD+ binding analysis, protein denaturation experiments, fluorescence lifetime analysis, steady-state anisotropy measurement, stopped flow fluorescence spectroscopy, and quantum yield determination. It was found that the conservative replacement of tryptophan residues with phenylalanine had little or no effect on the folded stability and enzyme activity of the PE24 protein. Dynamic quenching experiments indicated that when bound to the active site of the enzyme, the NAD+ substrate protected Trp-558 from solvent to a large extent but had no effect on the degree of solvent exposure for tryptophans 417 and 466. Also, upon substrate binding, the anisotropy of the Trp-417(W466F/W558F) protein showed the largest increase, followed by Trp-466(W417F/W558F), and there was no effect on Trp-558(W417F/W466F). Furthermore, the intrinsic tryptophan fluorescence exhibited the highest degree of substrate-induced quenching for the wild-type protein, followed in decreasing order by Trp-417(W466F/W558F), Trp-558(W417F/W466F), and Trp-466(W417F/W558F). These data provide evidence for a structural rearrangement in the enzyme domain near Trp-417 invoked by the binding of the NAD+ substrate.

Ligand-induced conformational changes in lactose repressor: a phosphorescence and ODMR study of single-tryptophan mutants.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on four single-tryptophan mutants of lac repressor protein from Escherichia coli: H74W/Wless, W201Y, Y273W/Wless, and F293W/Wless, where Wless represents a protein background containing the double mutation W201Y/W220Y. The single-tryptophan residues are located in the protein core region, either in the monomer-monomer interface of the tetrameric protein or in the region of the inducer binding cleft. Inducer binding elicits large changes in the energy (0,0-band wavelength shifts) and zero-field splitting energies (ZFS) of the triplet states for each of the mutant proteins except W201Y which exhibits more modest effects. F293W/Wless exists in two distinguishable conformations, only one of which appears to be sensitive to the presence of inducer. These effects of inducer binding can be attributed to a conformational change that alters specific polar interactions that occur at each affected tryptophan site. Changes in the tryptophan triplet state indicator depend on the existence of specific polar interactions that are altered by local atomic relocations.

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

Membrane topography of the T domain of diphtheria toxin probed with single tryptophan mutants.

The membrane insertion and translocation of diphtheria toxin, which is induced in vivo by low pH, is thought to be directed by the hydrophobic alpha-helices of its transmembrane (T) domain. In this study the structure of membrane-associated T domain was examined. Site-directed mutants of the T domain with single Trp residues were prepared at the two naturally occurring positions, 206 (near the N-terminal end of helix TH1) and 281 (within helix TH5), as well as at three residues in helix TH9, in which the substitutions F355W (near the N-terminal end of TH9), I364W (close to the center of TH9), and Y375W (near the C-terminal end of TH9) were made. All these mutants were found to undergo the low-pH-induced conformational change observed with wild-type T domain and insert into model membranes at low pH. The location of Trp residues relative to the lipid bilayer was characterized in model membrane vesicles by fluorescence emission and by quenching with nitroxide-labeled phospholipids. In TH9, residue 375 was shallowly inserted, residue 364 deeply inserted, and residue 355 located at an intermediate depth. Residues 206 and 281 exhibited moderately deep insertion. It was also found, in agreement with our previous study using bimane-labeled protein (Wang et al. (1997) J. Biol. Chem. 272, 25091-25098), that TH9 switches from a relatively shallowly inserted state to a more deeply inserted state when the concentration of the T domain in the membrane is increased or the thickness of the membrane bilayer is decreased. In particular, the depth of residue 355 was found to increase under the conditions giving deeper insertion. In contrast, residue 375 remained shallowly located in both states, as predicted from its location on the polar C-terminus of TH9. It is concluded that TH1 and TH5 insert into the lipid bilayer in both T domain conformations. In addition, Trp depths suggest that even in the shallowly inserted state there is a significant degree of insertion of TH9. These results suggest regions of the T domain in addition to the hydrophobic TH8/TH9 hairpin insert into membranes. Models for the structure of the membrane-inserted T domain are discussed.

Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.

Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of fructose-2,6-bisphosphate also significantly increased the anisotropy of W15. In the case of fructose-6-phosphate binding, the increased anisotropy was shown to be due to a restriction of the tryptophan residue's local mobility in the presence of bound ligand, which suggests that the N-terminus is located near the kinase active site. These increases in anisotropies were used to estimate the dissociation constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/- 0.3 microM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.

Steady state and time-resolved fluorescence study of residual structures in an unfolded form of yeast phosphoglycerate kinase.

A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two tryptophan residues in an unfolded form of yeast phosphoglycerate kinase [Garcia, P., et al. (1995) Biochemistry 34, 397-404]. In this paper, we present a more detailed characterization of these residual structures, through the study of two single tryptophan-containing mutants of yPGK, W333F and W308Y. Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures. On the other hand, the compactness and dynamics of the protein matrix were probed using tryptophan fluorescence quenching by acrylamide at various denaturant concentrations. Taking into consideration the changes in sample viscosity induced by addition of guanidinium chloride made feasible the use of this technique during the denaturation process. These different approaches have shown that the residual structures around tryptophan 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding tryptophan 333. Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy. In the native protein, tryptophan buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas tryptophan that is partially accessible to the solvent, W308, has a lifetime close to 0. 5 ns. The time-resolved tryptophan fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single tryptophan mutants. The significance of minor long lifetime components is discussed for the two tryptophan residues. This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of tryptophan 333 is modified for denaturant concentrations lower than those for tryptophan 308.

Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate.

A monomeric version of the dimeric tryptophan repressor from Escherichia coli, L39E TR, has previously been shown to resemble a transient intermediate that appears in the first few milliseconds of folding [Shao, X., Hensley, P., and Matthews, C. R. (1997) Biochemistry 36, 9941-9949]. In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the monomeric form with those of the native, dimeric form. The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Trp99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circular dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C. Coincident normalized transitions show that the urea denaturation process for each single-tryptophan mutant follows a two-state model involving monomeric native and unfolded forms. The free energies at standard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer. Fluorescence and near-UV circular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well-structured environments that are distinctively different from those found in their dimeric counterparts. Acrylamide quenching experiments show that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure. Measurements of the steady-state anisotropies of Trp19/L39E and Trp99/L39E TR demonstrate that the motions of both tryptophan side chains are restricted in the folded conformation. On the basis of these data, it can be concluded that this monomeric form of the tryptophan repressor adopts a well-folded, stable conformation with nonnative tertiary structure. When combined with previous results, the current findings demonstrate that the development of higher order structure during the folding of this intertwined dimer does not follow a simple hierarchical model.

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka∼1013M−1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 Å were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.

Effects of metal binding affinity on the chemical and thermal stability of site-directed mutants of rat oncomodulin

Tryptophan fluorescence was used to study the stability and unfolding behavior of several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM); F102W, Y57W, Y65W and the engineered protein CDOM33 which had the 12 residues of the CD loop replaced with a more potent metal binding site. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo<Ca 2+<Tb 3+. During thermal denaturation, the transition midpoints ( T un) of Y65W was the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which acted through the disruption of hydrogen bonds at the C-terminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinate number (i.e., Tb 3+) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability.

Roles of G(o)alpha tryptophans in GTP hydrolysis, GDP release, and fluorescence signals.

Single tryptophan mutants of a histidine-tagged G(o)alpha (W132F and W212F) were prepared to examine the functional and spectroscopic role of tryptophan in G(o)alpha. The mutants bound GTP gamma S with high affinity and showed only modest changes in GDP affinity. GTP gamma S-stimulated intrinsic fluorescence changes were completely abolished by removal of W212 but were not affected by elimination of W132. In contrast, both W132 and W212 contributed to the fluorescence signal from binding of methylanthraniloyl-GTP gamma S (mGTP gamma S). W132F and W212F mutants showed 57% and 34% of the mGTP gamma S fluorescence change of wild type (WT), respectively. The decreased fluorescence signals were not due to reduced activation of the W212F protein by nucleotide as protection from tryptic digestion was unchanged. The kinetics of nucleotide binding and hydrolysis were also altered in both mutants. GDP dissociation was slower (0.14 min-1) for W132F and faster (0.54 min-1) for W212F than for WT (0.25 min-1). As expected, the steady-state Vmax for GTPase was lower for W132F, but surprisingly it was also lower for W212F despite faster GDP release. Single turnover kinetics revealed a lower kcat for W212F (0.52 min-1) compared to WT (1.39 min-1) and W132F (1.0 min-1). Thus, W212 in G(o)alpha makes a dominant contribution to both intrinsic and extrinsic fluorescence signals upon alpha subunit activation. In addition, both tryptophans modulate the kinetics of nucleotide binding and hydrolysis.