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PubMed1999

Analysis of the dynamics of rhizomucor miehei lipase at different temperatures.

The dynamics of Rhizomucor miehei lipase has been studied by molecular dynamics simulations at temperatures ranging from 200-500K. Simulations carried out in periodic boundary conditions and using explicit water molecules were performed for 400 ps at each temperature. Our results indicate that conformational changes and internal motions in the protein are significantly influenced by the temperature increase. With increasing temperature, the number of internal hydrogen bonds decreases, while surface accessibility, radius of gyration and the number of residues in random coil conformation increase. In the temperature range studied, the motions can be described in a low dimensional subspace, whose dimensionality decreases with increasing temperature. Approximately 80% of the total motion is described by the first (i) 80 eigenvectors at T=200K, (ii) 30 eigenvectors at T=300K and (iii) 10 eigenvectors at T=400K. At high temperature, the alpha-helix covering the active site in the native Rhizomucor miehei lipase, the helix at which end the active site is located, and in particular, the loop (Gly35-Lys50) show extensive flexibility.

Lipase↗

PubMed2004

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis.

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

Amino Acid Sequence↗

PubMed2003

Characterization of molten globule state of fetuin at low pH.

Effect of pH over a range of 0.8-10 on bovine serum fetuin (BSF) was observed by far and near-UV circular dichroism (CD) spectroscopy, intrinsic tryptophan fluorescence and ANS fluorescence measurements. It has been reported earlier by our group that a molten globule (MG) state exists in alpha-chymotrypsinogen [Biochim. Biophys Acta 1481 (2000) 229] and stem bromelain [Eur. J. Biochem. 269 (2002) 47] at low pH. In this paper we have shown the presence of an MG form of fetuin at low pH. The far-UV CD spectra showed the regain of secondary structure at pH 1.8 as compared to the complete loss of secondary structure in presence of 6 M GnHCl. Near-UV CD spectra showed disruption of tertiary structure at pH 1.8. Tryptophan fluorescence studies indicated that there is only a slight red shift in the wavelength emission maxima (lambdamax) of fetuin at low pH as compared to a significantly red-shifted spectrum of the completely unfolded state in 6 M GnHCl, indicating that the tryptophan environment of fetuin at low pH resembles more the native form. ANS binding experiments also showed an enhancement in ANS binding with decrease in pH up to 1.8. ANS binding was absent at pH 7 and in the presence of 6 M GnHCl. Fluorescence quenching experiments were also performed with acrylamide, cesium chloride and potassium iodide. The quenching of tryptophan fluorescence by the three different quenchers indicates that low pH induces a conformational change in protein, making the tryptophan residue less accessible to solvent. This suggests that a more compact structure exists at low pH. The results, being in accordance with far-UV CD and fluorescence studies, imply the presence of MG state of fetuin at low pH. As studied by fluorescence spectroscopy, denaturation of fetuin at low pH was found to be reversible.

Animals↗

PubMed2003

Hypochlorite action on plasma fibronectin promotes its extended conformation in complexes with antibodies.

We investigated the influence of hypochlorite (HOCl/OCl-) on plasma fibronectin (Fn) aggregation and examined an affinity of Fn aggregates to Fn specific antibodies. Human plasma Fn HOCl/OCl(-)-mediated modification was monitored with differential OD method and with measurements of tryptophan fluorescence followed by acrylamide quenching of tryptophan emission. Antibody fibronectin complex formation was examined in ELISA systems with chemiluminescence (CL) detection. Results were expressed as an average of three experiments performed in triplicate. Fn aggregation/fragmentation was monitored with dynamic light scattering method. It was showed that HOCl/OCl- mediated chlorination promotes Fn aggregation/fragmentation with concomitant oxidation of tryptophan moieties and dichlorotyrosine formation. Quenching experiments revealed that in chlorinated Fn the percentage of intact tryptophan moieties buried in the hydrophobic Fn core increases as compared to unchlorinated Fn. In general, ELISA experiments showed that chlorination of plasma Fn diminished the number of available epitopes but for lower HOCl/OCl- concentrations (1-2 mM) the reverse effect is observed--the number of accessible fibronectin epitopes is increased when Fn adopts extended conformation in complex with antibody. Our results suggest that HOCl/OCl(-)-mediated plasma Fn chlorination leads to the formation of soluble aggregates and is followed by refolding processes. Fn chlorination with low doses of HOCl/OCl- promotes extended Fn conformation which in turn increases affinity toward specific antibodies and may promote Fn-IgG cluster formation. Thus it seems possible that mildly chlorinated plasma Fn promotes formation of IgG clusters which in turn may activate neutrophils.

Acrylamide↗

PubMed1983

Conformational analysis of the calcium-antagonist gallopamil.

Conformational analysis of gallopamil was performed in order to gain insight into the molecular determinant of its calcium-antagonistic property. Whereas the neutral form of gallopamil was characterized by a single, largely predominant configuration, the protonated form of the drugs yielded several conformers, some of which were characterized by a readily accessible ionized site. The capacity of gallopamil to inhibit ionophore-mediated calcium translocation in a two-phase bulk system was inversely related to the pH of the aqueous phase. These findings indicate that the capacity of gallopamil to interfere with the transport of cations is critically dependent on the availability of a protonated configuration of the drug.

Calcimycin↗

PubMed2000

Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer.

The kinetics of O2*- reaction with semi-oxidized tryptophan radicals in lysozyme, Trp*(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp*(Lyz) radicals were formed by Br2*- oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 x 10(8) M(-1) s(-1); but drops to 8 x 10(7) M(-1) s(-1) or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp*(Lyz) + Trp*(Lyz) recombination reaction is about (4-7 x 10(6) M(-1) s(-1)) at/or below pH 6.2 then drops to 1.3-1.6 x 10(6) M(-1) s(-1) at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semi-oxidized Trp* residues upon Br2*- reaction. The kinetics of Trp*(Lyz) with ascorbate, a reducing species rather larger than O2*- have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals--leading to the repair of the semi-oxidized Trp*(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp*(Lyz) + O2*- reaction, as is most of the Trp*(Lyz) + Trp*(Lyz) reaction. However, the kinetic behavior of Trp*(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O2*-. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O2*- reactivity. To our knowledge, this is the first report on the kinetics of a protein-O2*- reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.

Anions↗

PubMed1988

Surface, subunit interfaces and interior of oligomeric proteins.

The solvent-accessible surface area (As) of 23 oligomeric proteins is calculated using atomic co-ordinates from high-resolution and well-refined crystal structures. As is correlated with the protein molecular weight, and a power law predicts its value to within 5% on average. The accessible surface of the average oligomer is similar to that of monomeric proteins in its hydropathy and amino acid composition. The distribution of the 20 amino acid types between the protein surface and its interior is also the same as in monomers. Interfaces, i.e. surfaces involved in subunit contacts, differ from the rest of the subunit surface. They are enriched in hydrophobic side-chains, yet they contain a number of charged groups, especially from Arg residues, which are the most abundant residues at interfaces except for Leu. Buried Arg residues are involved in H-bonds between subunits. We counted H-bonds at interfaces and found that several have none, others have one H-bond per 200 A2 of interface area on average (1 A = 0.1 nm). A majority of interface H-bonds involve charged donor or acceptor groups, which should make their contribution to the free energy of dissociation significant, even when they are few. The smaller interfaces cover about 700 A2 of the subunit surface. The larger ones cover 3000 to 10,000 A2, up to 40% of the subunit surface area in catalase. The lower value corresponds to an estimate of the accessible surface area loss required for stabilizing subunit association through the hydrophobic effect alone. Oligomers with small interfaces have globular subunits with accessible surface areas similar to those of monomeric proteins. We suggest that these oligomers assemble from preformed monomers with little change in conformation. In oligomers with large interfaces, isolated subunits should be unstable given their excessively large accessible surface, and assembly is expected to require major structural changes.

Amino Acids↗

PubMed2000

A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex.

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.

Antibodies, Monoclonal↗

PubMed1995

The conformation of the active site of myosin probed using mant-nucleotides.

Changes in the conformation of the active site of myosin subfragment-1 (S1) may be linked to the production of force during the powerstroke. We probed the conformation of the nucleotide pocket by measuring the solvent accessibility of bound mant-nucleotides. Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent phase quencher acrylamide. The fluorescent mant moiety is attached to the ribose and is located near the outside of the pocket where it is likely to be sensitive to opening of the pocket. MantADP was highly protected from the quencher when bound to the active site of S1. A similar degree of protection was also observed for mantATP during steady-state hydrolysis by S1, and for mantADP bound to acto-S1 or to myosin in myofibrils. Assuming that S1-mantATP and actoS1-mantADP represent states at the beginning and the end of the powerstroke, respectively, we conclude that the myosin nucleotide pocket does not undergo a large conformational change during the powerstroke. However, the high degree of protection seen for mant-nucleotides is not easily explained by the open structure of the nucleotide pocket in the S1-nucleotide complex observed by x-ray diffraction.

Acrylamide↗

PubMed1985

A spin label study of the ionic strength dependent conformational change in the human Ia molecule.

Purified human Ia molecules were labelled with maleimide or isothiocyanate spin labels or by reacting "TEMPAMINE" spin label with the neuraminic acid of their carbohydrate residues. It was found that increasing the ionic strength from 0.05 to 0.75 markedly increased the dipolar interaction between the maleimide-attached labels, but no effect was found of ionic strength or motion on dipolar interaction with the other two labels. The effect of increasing ionic strength could be blocked by the prior addition of Ia-specific antibody, but could not be reversed by the addition of antibody after ionic strength was increased. These findings complement an earlier finding that increasing ionic strength over the range 0.05-0.75 has an inhibitory effect on the combination of Ia with its antibody. Because the maleimide spin labels attach predominantly to SH groups it is suggested that increasing ionic strength causes conformational changes in the immunoglobulin loop region which alter the accessibility of the Ia antibody-binding site.

Antibody Specificity↗

PubMed2004

Influence of a fluorobenzene nucleobase analogue on the conformational flexibility of RNA studied by molecular dynamics simulations.

Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-beta-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-beta-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable 'base-paired' geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.

Adenine↗

PubMed1986

Chromatin structure and induction-dependent conformational changes of human interferon-beta genes in a mouse host cell.

Multiple copies of a human interferon-beta gene introduced into a mouse host cell line can be activated by induction with double-stranded RNA. Several induction-dependent changes of the chromatin structure could be traced by mapping techniques using four different agents [DNase I, micrococcus nuclease, bromoacetaldehyde and methidiumpropyl-EDTA X iron(II)]. Our data show that all copies of the interferon gene have adopted a very similar conformation in the host cell and respond to the inducing stimulus in a highly synchronous fashion. Detailed induction-specific changes were observed best with the chemical reagents which disclose a specific hypersensitive site within a sequence that has been shown to be required for the induction process (around position -80) and three other regions which, in addition to the transcribed region itself, gain single-strand character by an auxiliary process which can be mimicked by the addition of butyrate to the medium and may therefore involve histone hyperacetylation. Six discrete 'phased' nucleosomes are present upstream from the gene and are modulated by induction. At least four nucleosomes are located downstream. The interferon genes are largely protected from micrococcus nuclease in the inactive state. Gene activation increases access to micrococcus nuclease and DNase I indicating gross conformational changes on a higher level of chromatin structure.

Animals↗

PubMed1989

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model.

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗

PubMed2004

Probing flexibility and "induced-fit" phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations.

Aldose reductase is a promising target for the treatment of diabetic complications, and as such, has become the focus of various drug design projects. As revealed by a survey of available crystal structures, the protein shows pronounced induced-fit effects upon ligand binding. Although helping to explain the enzyme's substrate promiscuity, phenomena of this kind are still responsible for significant complications in structure-based design efforts directed to aldose reductase. Accordingly, a deeper understanding of the principles governing conformational alterations in this enzyme would be of utmost practical importance. As a first step in addressing this issue, molecular dynamics (MD) simulations have been carried out. The ultrahigh resolution crystal structure of aldose reductase complexed with inhibitor IDD594 served as ideal starting point for a set of different simulations of nanosecond time scale: the native complexed state with bound inhibitor, the uncomplexed state (after removal of the inhibitor) at standard temperature, and the uncomplexed state at elevated temperature. The reference simulation of the complex exhibits extraordinary stability of the overall fold, whereas two distinct conformational substates are found for the binding-site region. In contrast, already at standard temperature pronounced changes are observed in the binding region during the simulation of the uncomplexed state. Leu300, for example, closes the access to the pocket opened by IDD594. On the other hand, conformations around the catalytic site are highly conserved, with the His110-Tyr48-NADP+ orientation being stabilized by a water molecule. Detailed analysis of the trajectories allows to reveal a set of distinct conformational substates that may prove useful as alternative structural templates in virtual screening for new aldose reductase inhibitors.

Acetates↗

PubMed1984

Two human colon tumor cell lines with similar nuclease sensitivities have different ethidium bromide binding characteristics.

Chromatin from two human colon adenocarcinoma cell lines (HT-29 and LoVo) showed similar digestion kinetics when sensitivities to DNase I and micrococcal nuclease were examined. Chromatin conformations were probed by examining the binding of ethidium bromide. A Scatchard plot revealed that both chromatins bound the same amount of ethidium bromide per mole of DNA, but the DNA from LoVo cells was more accessible to the intercalator. The results indicate that differences in chromatin conformation are not necessarily accompanied by different nuclease sensitivities.

Adenocarcinoma↗

PubMed2005

Dynamics and mechanism of the Tanford transition of bovine beta-lactoglobulin studied using heteronuclear NMR spectroscopy.

The Tanford transition is a conformational change of bovine beta-lactoglobulin (betaLG) occurring at around pH 7, identified originally on the basis of optical rotatory dispersion and the accessibility of a thiol group. X-ray analysis has suggested that a conformational change to the EF-loop is responsible for the Tanford transition, with the loop closing the hydrophobic cavity of the beta-barrel of the betaLG molecule below pH 7 and flipping to open the cavity above pH 7. To clarify the dynamics of this conformational change, NMR measurements were made at neutral pH. Since severe signal broadening due to monomer-dimer equilibrium prevented NMR measurements of wild-type betaLG at neutral pH, we searched for optimal sample conditions, finding that a disulfide bond-linked dimer of the mutant A34C gives an HSQC spectrum without signal broadening. The HSQC and CD spectra indicated that in overall conformation A34C is similar to wild-type betaLG, suggesting that the A34C dimer is a good model with which to study the structure and dynamics of the wild-type at neutral pH. The pH-dependent HSQC signal changes and Lipari-Szabo type relaxation analyses of the A34C dimer revealed that the conformational change to the EF-loop occurs above pH 7. We observed two types of motions in the EF-loop region; relatively fast (micro- to milliseconds) and slow (milliseconds or slower) conformational exchanges of the residues located in the hinge and top of the EF-loop regions, respectively. Furthermore, the GH-loop adjacent to the EF-loop exhibited conformational change at a pH slightly lower than that at which the EF-loop motions occurred. From these observations, we propose a three-step mechanism of conformational change in the EF-loop leading to the Tanford transition, in which the GH-loop conformational change, the cleavage of the hydrogen bonds at the hinge, and the flip of the EF-loop occur sequentially.

Animals↗

PubMed2002

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

Antibody Affinity↗

PubMed2004

Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor.

Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by alpha-amino-n-butyric acid (Abu) while retaining the disulfide between Cys 14 and 38; the resultant variant is termed [14-38](Abu). Two new analogues with modifications in the beta-turn, P26D27[14-38](Abu) and N26G27K28[14-38](Abu), are compared to partially folded [14-38](Abu), as well as to [R](Abu), the unfolded protein with all six Cys residues replaced by Abu. Structural features of the new analogues of [14-38](Abu) have been determined by circular dichroism (CD), one-dimensional (1)H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Both analogues are more disordered than the parent [14-38](Abu), but while P26D27[14-38](Abu) has a small population of native-like conformations observed by NMR, no ordered structure is detected for N26G27K28[14-38](Abu). Trypsin inhibition assays were carried out using a modified rat trypsin, C191A/C220A, that minimizes cleavage of unfolded peptides. Both [14-38](Abu) and P26D27[14-38](Abu) significantly inhibit modified trypsin. N26G27K28[14-38](Abu) has low but measurable inhibitor activity, while [R](Abu) has no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by [14-38](Abu) and by both variants but not by [R](Abu). We conclude that partially folded ensembles of BPTI, even those with little or no CD- or NMR-detectable structure, contain minor populations of native-like conformations. Partially folded [14-38](Abu) and both variants, as well as [R](Abu), have enhanced negative ellipticity in CD spectra acquired in the presence of the osmolyte trimethylamine N-oxide (TMAO). TMAO-induced structure is formed cooperatively, as indicated by thermal unfolding curves. Inhibitor activity as a function of TMAO concentration implies that the osmolyte-induced structure is native-like for [14-38](Abu) and P26D27[14-38](Abu) and is probably native-like for N26G27K28[14-38](Abu). [R](Abu) also shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.

Amino Acid Substitution↗