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One popular model for protein folding, the framework model, postulates initial formation of secondary structure elements, which then assemble into the native conformation. However, short peptides that correspond to secondary structure elements in proteins are often only marginally stable in isolation. A 33-residue peptide (GCN4-p1) corresponding to the GCN4 leucine zipper folds as a parallel, two-stranded coiled coil [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T.A. (1991) Science 254, 539-544]. Deletion of the first residue (Arg 1) results in local, N-terminal unfolding of the coiled coil, suggesting that a stable subdomain of GCN4-p1 can form. N- and C-terminal deletion studies result in a 23-residue peptide, corresponding to residues 8-30 of GCN4-p1, that folds as a parallel, two-stranded coil with substantial stability (the melting temperature of a 1 mM solution is 43 degrees C at pH 7). In contrast, a closely related 23-residue peptide (residues 11-33 of GCN4-p1) is predominantly unfolded, even at 0 degrees C, as observed previously for many isolated peptides of similar length. Thus, specific tertiary packing interactions between two short units of secondary structure can be energetically more important in stabilizing folded structure than secondary structure propensities. These results provide strong support for the notion that stable, cooperatively folded subdomains are the important determinants of protein folding.

Proteins have many distinct tertiary folds (Richardson, J. S. (1981) Adv. Prot. Chem. 34, 167-339). The term tertiary fold refers to the spatial organization of secondary structure elements (alpha-helices and beta-strands). It is not known when, in the process of protein folding, a native tertiary fold emerges. Here, we show that the helical domain of human alpha-lactalbumin, in isolation, forms a molten globule with the same overall tertiary fold as that found in intact alpha-lactalbumin. Formation of this native-like fold does not require extensive, specific side-chain packing. Our results suggest that much of the information transfer from one-dimension to three-dimensions has occurred at the molten globule stage of protein folding.

Health care organizations across the nation have begun instituting quality improvement programs in order to meet the demands of improving service and clinical quality in health care. The implementation process requires time, effort, and money in order for the health care organizations to learn who their customers are and what they want. The results, which sometimes are not recognized until after a two- to three-year period, include reduced costs by cutting waste and complexity and by doing things right the first time. Still, there are many challenges in adapting total quality management (TQM) to public organizations such as the health care industry. This article discusses the founding principles of TQM and how they are applied in implementing TQM to enhance quality in health care organizations. This article also forecasts the future of TQM implementation in public health care.

Bovine pancreatic trypsin inhibitor (BPTI) unfolds upon reduction of its three disulfide bonds. A recombinant model of the reduced state of BPTI, called [R]Ala, in which all six Cys residues are replaced with Ala, has been expressed in Escherichia coli. 1H nuclear magnetic resonance spectroscopy shows that [R]Ala does not contain stable secondary structure. Some chemical shift dispersion exists, however, suggesting the existence of non-random interactions in [R]Ala. In particular, the side-chain protons of Ile19 resonate upfield of those of Ile18. This observation was investigated using an eight residue peptide model, P17-24, corresponding to residues 17 to 24 of BPTI. The non-random chemical shift dispersion of the Ile residues observed in [R]Ala also occurs in P17-24, indicating that P17-24 contains interactions that are similar to those found in the corresponding region of [R]Ala. The only interresidue nuclear Overhauser effects observed in P17-24 are between the ring protons of Tyr21 and the gamma CH3 group of Ile19, indicating that these protons are in close proximity. Substitution of Tyr21 by Ala in P17-24 results in the loss of the chemical shift dispersion of the Ile resonances, suggesting that the upfield shifts of the Ile19 resonances are due to ring current shifts arising from the proximity of Tyr21. Collectively, these results suggest that the side-chain of Ile19 is positioned at least some of the time above the plane of the aromatic ring of Tyr21. We conclude that these two residues participate in a hydrophobic cluster in P17-24 and in the denatured state of BPTI.

Several model systems have been used to evaluate the alpha-helical propensities of different amino acids. In contrast, experimental quantitation of beta-sheet preferences has been addressed in only one model system, a zinc-finger peptide. Here we measure the relative propensity for beta-sheet formation of the twenty naturally occurring amino acids in a variant of the small, monomeric, beta-sheet-rich, IgG-binding domain from protein G. Amino-acid substitutions were made at a guest site on the solvent-exposed surface of the beta-sheet. Several criteria were used to establish that the mutations did not cause significant structural changes: binding to the Fc domain of IgG, calorimetric unfolding and NMR spectroscopy. Characterization of the terminal stabilities of these proteins leads to a thermodynamic scale for beta-sheet propensities that spans a range of approximately 2 kcal mol-1 for the naturally occurring amino acids, excluding proline. The magnitude of the differences suggests that beta-sheet preferences can be important determinants of protein stability.

Coiled-coil sequences in proteins consist of heptad repeats containing two characteristic hydrophobic positions. The role of these buried hydrophobic residues in determining the structures of coiled coils was investigated by studying mutants of the GCN4 leucine zipper. When sets of buried residues were altered, two-, three-, and four-helix structures were formed. The x-ray crystal structure of the tetramer revealed a parallel, four-stranded coiled coil. In the tetramer conformation, the local packing geometry of the two hydrophobic positions in the heptad repeat is reversed relative to that in the dimer. These studies demonstrate that conserved, buried residues in the GCN4 leucine zipper direct dimer formation. In contrast to proposals that the pattern of hydrophobic and polar amino acids in a protein sequence is sufficient to determine three-dimensional structure, the shapes of buried side chains in coiled coils are essential determinants of the global fold.

Background: The leucine zipper is a protein structural motif involved in the dimerization of a number of transcription factors. We have previously shown that peptides corresponding to the leucine-zipper region of the Fos and Jun oncoproteins preferentially form heterodimeric coiled coils, and that simple principles involving electrostatic interactions are likely to determine the pairing specificity of coiled coils. A critical test of these principles is to use them as guidelines to design peptides with desired properties., Results: Based on studies of the Fos, Jun and GCN4 leucine zippers, we have designed two peptides that are predominantly unfolded in isolation but which, when mixed, associate preferentially to form a stable, parallel, coiled-coil heterodimer. To favor heterodimer formation, we chose peptide sequences that would be predicted to give destabilizing electrostatic interactions in the homodimers that would be relieved in the heterodimer. The peptides have at least a 10(5)-fold preference for heterodimer formation, and the dissociation constant of the heterodimer in phosphate-buffered saline is approximately 30 nM at pH 7 and 20 degrees C. Studies of the pH and ionic strength dependence of stability confirm that heterodimer formation is favored largely as a result of electrostatic destabilization of the homodimers., Conclusions: Our successful design strategy supports previous conclusions about the mechanism of interaction between the Fos and Jun oncoproteins. These results have implications for protein design, as they show that it is possible to design peptides with simple sequences that have a very high preference to pair with one another. Finally, these sequences with 'Velcro'-like properties may have practical applications, including use as an affinity reagent, in lieu of an epitope tag, or as a way of bringing together two molecules in a cell.

Protein disulphide isomerase (PDI) is a highly abundant and ubiquitous eukaryotic protein that is essential for viability in yeast. Although PDI is thought to catalyse disulphide bond formation and isomerization during protein biosynthesis, PDI has been found previously to have only moderate effects (approximately 25-fold) on the rate of oxidative folding of proteins in vitro. In addition, PDI has been implicated in several apparently unrelated cellular functions. For example, PDI is the beta-subunit of prolyl 4-hydroxylase and is part of the triglyceride transfer complex. The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) is slow and inefficient in vitro. Here we report that PDI increases by a factor of 3,000-6,000 the rates of folding of kinetically trapped BPTI folding intermediates, in which native structure impedes disulphide bond formation. By contrast, PDI has only small effects on the rate of disulphide bond formation in intermediates that are oxidized readily in the absence of PDI. These results suggest that an important function of PDI is to catalyse disulphide bond formation and rearrangements within kinetically trapped, structured folding intermediates.

In the endoplasmic reticulum (ER) of cultured porcine thyrocytes, newly synthesized thyroglobulin (Tg, the precursor in thyroid hormone synthesis) initially forms protein aggregates, which are dissolved into monomers and then assembled to dimers, before intracellular transport and secretion. However, studies suggest that in different physiological states and in different cells, folding efficiency in the ER may vary; with this in mind we have set out to further characterize the phenomenon of nascent Tg aggregation. In primary cultured thyrocytes, fresh thyroid follicular tissue (of porcine and rat origin), and the FRTL-5 cell line, nascent Tg appears transiently aggregated with mispaired, interchain disulfide linkages. Using a cell lysis procedure that maximally inhibits proteolysis as well as artifactual disulfide formation, Tg aggregates of M(r) > or = 2,000,000 can be stably isolated by gel filtration. Furthermore, stimulation with thyrotropin and other hormones that enhance Tg production may alter but does not eliminate formation of these aggregates. We conclude that transient disulfide-linked aggregation occurs normally during Tg folding in the ER of thyroid epithelial cells.

Influenza hemagglutinin (HA) undergoes a conformational change that induces viral fusion with the cellular membrane. The structure of HA in the fusogenic state is unknown. We have identified a sequence in HA that has a high propensity for forming a coiled coil. Surprisingly, this sequence corresponds to a loop region in the X-ray structure of native HA: the loop is followed by a three-stranded, coiled-coil stem. We find that a 36 residue peptide (LOOP-36), comprising the loop region and the first part of the stem, forms a three-stranded coiled coil. This coiled coil is extended and stabilized in a longer peptide, corresponding to LOOP-36 plus the residues of a preceding, short alpha helix. These findings lead to a model for the fusogenic conformation of HA: the coiled-coil stem of the native state extends, relocating the hydrophobic fusion peptide, by 100 A, toward the target membrane.

Electrostatic interactions in proteins are potentially quite strong, but these interactions are mitigated by the screening effects of water, ions, and nearby protein atoms. The early work of Kirkwood and Westheimer on small organic molecules showed that the extent of the screening may depend on whether charged or dipolar groups are involved. The dielectric and ionic screening of the interactions between the dipolar backbone amide groups of monomeric alpha helices and either (i) solvent-exposed charges or (ii) solvent-exposed dipoles at the amino terminus was measured. The dielectric screening effects are an order of magnitude greater for the backbone-charge interactions than for the backbone-dipole interactions, and the ionic strength dependence is substantially different in the two cases. These results suggest that interactions that involve the dipolar groups of proteins may be relatively more important for stability and function than is generally thought.

To understand how the endoplasmic reticulum (ER) of the thyrocyte remains flexible to physiologic changes in the load of exportable proteins, we have examined hormonally-induced increments in thyroglobulin (Tg) flux and pool size in the ER, the relationship between kinetics of Tg folding and ER export, and steady-state levels of molecular chaperones. Tg production was increased > or = 5-fold by chronic exposure to thyrotropin (TSH), and > or = 25-fold by exposure to a mixture of TSH, insulin, transferrin, and hydrocortisone (4H). In TSH-grown cells Tg assembly was accelerated, specifically involving early folding intermediates that lead to a compact monomer. Accelerated dissociation of nascent Tg from the binding protein, BiP, was observed in parallel. TSH exposure was accompanied by modest increases in ER chaperones as well as accelerated Tg export from the thyrocyte ER. However, in 4H-grown thyrocytes, although there were further increases in ER chaperones, monomer maturation was slowed and the association between nascent Tg and BiP was prolonged. Nevertheless, export from the ER remained accelerated, indicating that exit from the ER must include other regulated steps that occur after the folding of exportable proteins. Thus, protein folding may not necessarily be the rate-limiting step in the export of newly synthesized proteins from the ER.

The in vitro folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been described previously in terms of the disulfide-bonded intermediates that accumulate during folding of the protein. Folding is slow, occurring in hours at pH 7.3, 25 degrees C. In addition, approximately half of the BPTI molecules become trapped as a dead-end, native-like intermediate. In vivo, BPTI is synthesized as a precursor protein that includes a 13 residue amino-terminal pro region. This pro region contains a cysteine residue. We find that, in vitro, both the rate of formation and the yield of properly folded BPTI are increased substantially in a recombinant model of pro-BPTI. The cysteine residue is necessary for this effect. Moreover, a single cysteine residue, tethered to the carboxy-terminal end of BPTI with a flexible linker of repeating Ser-Gly-Gly residues, is sufficient to assist in disulfide formation. Thus, the pro region appears to facilitate folding by providing a tethered, solvent-accessible, intramolecular thiol-disulfide reagent.

We have shown previously that during the oxidative folding of bovine pancreatic trypsin inhibitor only intermediates with native disulfide bonds are well populated. Nevertheless, these studies also confirmed the earlier conclusion [Creighton, T. E. (1977) J. Mol. Biol. 113, 275-293] that the rate-limiting transition in the kinetically preferred route for folding involves intramolecular disulfide bond rearrangements. Consequently, intermediates with nonnative disulfide bonds must form transiently during folding. Two specific nonnative species, denoted [30-51; 5-14] and [30-51; 5-38], in which numbers indicate residues participating in a disulfide bond, can be detected at low levels in kinetic folding experiments with bovine pancreatic trypsin inhibitor. By working with purified reversibly trapped intermediates, the role of these two nonnative species has been examined directly. These species are found to be in relatively rapid exchange with each other and with an initially formed native two-disulfide intermediate [30-51; 14-38]. Thus, the low abundance of the two nonnative species detected in kinetic folding experiments reflects primarily their low thermodynamic stability as compared to this native intermediate. To a small extent, these nonnative species form the productive native intermediate [30-51; 5-55], which is the immediate precursor to the native protein. However, an equal amount of [5-55; 14-38], a nonproductive dead-end intermediate, is also produced. Thus, the nonnative species detected during the folding of bovine pancreatic trypsin inhibitor are not committed to forming the productive native intermediate, nor do they serve to direct folding specifically toward a productive route.

The strengths of electrostatic interactions in biological molecules are difficult to calculate or predict because they occur in complicated, inhomogeneous environments. The electric field at the amino terminus of an alpha helix in water has been determined by measuring the shift in the absorption band for a covalently attached, neutral probe molecule with an electric dipole moment difference between the ground and excited electronic states (an internal Stark effect). The field at the interface between the helix and the solvent is found to be an order of magnitude stronger than expected from the dielectric properties of bulk water. Furthermore, although the total electric dipole moment of the helix increases with length, the electric field at the amino terminus does not.

NMR experiments show that a stable complex can be formed between a 14-base-pair oligonucleotide and a disulfide-bonded dimer of a peptide containing 27 residues of the basic region of the yeast transcriptional activator GCN4; the complex is in slow exchange on the NMR time scale. In contrast, a nonspecific complex is in fast exchange on the NMR time scale. DNase I footprinting experiments show that dimers of peptides containing as few as 20 residues of GCN4 bind DNA with sequence specificity similar to that of the intact protein. Circular dichroism experiments suggest that specific binding involves only 15 residues, corresponding to residues 231-245 of GCN4, in an alpha-helical conformation. These results limit substantially the region of GCN4 involved in sequence-specific DNA contacts and provide a uniquely simple model for studying protein-DNA interactions in detail.

Because of its unusual length, nascent thyroglobulin (Tg) requires a long time after translocation into the endoplasmic reticulum (ER) to assume its mature tertiary structure. Thus, Tg is an ideal molecule for the study of protein folding and export from the ER, and is an excellent potential substrate for molecular chaperones. During the first 15 min after biosynthesis, Tg is found in transient aggregates with and without interchain disulfide bonds, which precede the formation of free monomers (and ultimately dimers) within the ER. By immunoprecipitation, newly synthesized Tg was associated with the binding protein (BiP); association was maximal at the earliest chase times. Much of the Tg released from BiP by the addition of Mg-ATP was found in aggregates containing interchain disulfide bonds; other BiP-associated Tg represented non-covalent aggregates and unfolded free monomers. Importantly, the immediate precursor to Tg dimer was a compact monomer which did not associate with BiP. The average stoichiometry of BiP/Tg interaction involved nearly 10 BiP molecules per Tg molecule. Cycloheximide was used to reduced the ER concentration of Tg relative to chaperones, with subsequent removal of the drug in order to rapidly restore Tg synthesis. After this treatment, nascent Tg aggregates were no longer detectable. The data suggest a model of folding of exportable proteins in which nascent polypeptides immediately upon translocation into the ER interact with BiP. Early interaction with BiP may help in presenting nascent polypeptides to other helper molecules that catalyze folding, thereby preventing aggregation or driving aggregate dissolution in the ER.

In the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) at neutral pH, only two one-disulfide intermediates accumulate to a significant extent, namely [5-55] and [30-51]. In this paper we describe a recombinant model of [5-55], designated [5-55]Ala, which was made by replacing the cysteine residues not involved in the disulfide bond with alanine. As judged by two-dimensional NMR, [5-55]Ala folds into essentially the same conformation as native BPTI. Moreover, like native BPTI, [5-55]Ala inhibits trypsin stoichiometrically. Thus, the disulfide-bonded intermediate [5-55] corresponds not to a partially folded protein folding intermediate but rather to an essentially completely folded protein. This conclusion provides an explanation for many of the thermodynamic and kinetic properties of [5-55] in the folding pathway of BPTI.

We describe a series of experiments that aimed to establish whether nuclease activity is actually associated with diphtheria toxin (DTx) and its A subunit (DTA), as we originally reported (M. P. Chang, R. L. Baldwin, C. Bruce, and B. J. Wisnieski, Science 246:1165-1168, 1989). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.

Fos and Jun, the protein products of the nuclear proto-oncogenes c-fos and c-jun, associate preferentially to form a heterodimer that binds to DNA and modulates transcription of a wide variety of genes in response to mitogenic stimuli. Both Fos and Jun contain a single leucine zipper region. Previous studies have shown that the leucine zippers of Fos and Jun are necessary and sufficient to mediate preferential heterodimer formation. The leucine zipper regions from Fos and Jun are also known to fold autonomously, most likely as two-stranded, parallel coiled coils. We show here that 8 amino acids from Fos and from Jun are sufficient to mediate preferential heterodimer formation in a background of the GCN4 leucine zipper sequence. Using pH titration and amino acid replacements, we also show that destabilization of the Fos homodimer by acidic residues provides a major thermodynamic driving force for preferential heterodimer formation.

The two-stranded coiled-coil motif, which includes leucine zippers, is a simple protein structure that is well suited for studies of helix-helix interactions. The interaction between helices in a coiled coil involves packing of "knobs" into "holes", as predicted by Crick in 1953 and confirmed recently by X-ray crystallography for the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T. (1991) Science 254, 539]. A striking periodicity, extending over six helical turns, is observed in the rates of hydrogen-deuterium exchange for amide protons in a peptide corresponding to the leucine zipper of GCN4. Protons at the hydrophobic interface show the most protection from exchange. The NMR chemical shifts of amide protons in the helices also show a pronounced periodicity which predicts a short H-bond followed by a long H-bond every seven residues. This variation was anticipated in 1953 by Pauling and is sufficient to give rise to a local left-handed superhelical twist characteristic of coiled coils. The amide protons that lie at the base of the "hole" in the "knobs-into-holes" packing show slow amide proton exchange rates and are predicted to have short H-bond lengths. These results suggest that tertiary interactions can lead to highly localized, but substantial, differences in stability and dynamics within a secondary structure element and emphasize the dominant nature of packing interactions in determining protein structure.

For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange. We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols. We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein. Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state. The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same. By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms.

The x-ray crystal structure of a peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4 has been determined at 1.8 angstrom resolution. The peptide forms a parallel, two-stranded coiled coil of alpha helices packed as in the "knobs-into-holes" model proposed by Crick in 1953. Contacts between the helices include ion pairs and an extensive hydrophobic interface that contains a distinctive hydrogen bond. The conserved leucines, like the residues in the alternate hydrophobic repeat, make side-to-side interactions (as in a handshake) in every other layer of the dimer interface. The crystal structure of the GCN4 leucine zipper suggests a key role for the leucine repeat, but also shows how other features of the coiled coil contribute to dimer formation.

Bovine pancreatic trypsin inhibitor (BPTI) continues to be the only protein for which a detailed pathway of folding has been described. Previous studies led to the conclusion that nonnative states are well populated in the oxidative folding of BPTI. This conclusion has broadly influenced efforts to understand protein folding. The population of intermediates present during the folding of BPTI has been reexamined by modern separation techniques. It was found that all well-populated folding intermediates contain only native disulfide bonds. These data emphasize the importance of native protein structure for understanding protein folding.

We have investigated the kinetics of folding and dimerization of newly synthesized thyroglobulin (Tg), the precursor protein in the manufacture of thyroid hormone. From the examination of lysates of pulse-labeled cultured thyrocytes by denaturing and nondenaturing gel electrophoresis, we have found that the earliest detectable form of nascent Tg is a transient aggregate, whose dissolution in vitro requires the addition of a reducing agent. In vivo, aggregate dissolution occurs with a t1/2 approximately 10 min at 37 degrees C. By 10 min after synthesis, monomeric Tg is first detectable in a conformationally unstable form. Dimeric Tg is formed thereafter (t1/2 approximately 30 min), but well before arrival of the protein in the medial Golgi (t1/2 approximately 2 h). Certain metabolic inhibitors permit dimerization yet block transport of the dimer to the Golgi. Thus, Tg dimerization occurs in a pre-Golgi compartment, and other steps after dimerization are likely to be important in the process of exit from the endoplasmic reticulum (ER). Further, aggregate dissolution, as well as dimerization, are inhibited significantly at 15 degrees C, indicating thermal sensitivity of Tg folding over and above effects on vesicular transport. Inhibitors of Tg iodination have no effect on Tg dimerization or Golgi arrival. Pretreatment of thyrocytes with thyroid-stimulating hormone substantially accelerates Tg flux through the ER, by increasing the amount, as well as the rate, of Tg transport, possibly at the expense of a small fraction of Tg that appears refractory to dimerization. Inhibition of N-linked glycosylation by tunicamycin causes a complete block in intracellular Tg transport by inducing the formation of biologically irreversible aggregates, suggesting that glycosylation of Tg serves to prevent denaturation of the secretory protein within the ER lumen.

Dimerization of the bZIP class of eukaryotic transcriptional control proteins requires a sequence motif called the leucine zipper. We have grown two distinct crystal forms of a 33-amino acid peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4. This peptide is known to form a dimer of parallel helices in solution. X-ray scattering from both crystal forms shows reflections that are diagnostic of coiled coils. The most notable reflections occur at approximately 5.2 A resolution and correspond to the pitch of helices in coiled coils. There is no diffraction maximum near 5.4 A, the characteristic pitch of straight helices. Our results provide direct evidence that the leucine zipper of GCN4 is a coiled coil.

A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization. This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4. When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues. In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.

A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization. A peptide model for the basic region of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide bond. The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4 degrees C. DNA binding is sequence-specific as judged by deoxyribonuclease I footprinting. Circular dichroism spectroscopy suggests that the peptide adopts a helical structure when bound to DNA. These results demonstrate directly that the GCN4 basic region is sufficient for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein dimerization. Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.

The disulphide-bonded intermediates that accumulate in the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) were characterized some time ago. Structural characterization of these intermediates would provide an explanation of the kinetically preferred pathways of folding for BPTI. When folding occurs under strongly oxidizing conditions, more than half the molecules become trapped in an intermediate, designated N*, which is similar to the native protein but lacks the 30-51 disulphide bond. We have tested the hypothesis that the precursor to N* is the one-disulphide intermediate [5-55], which contains the most stable disulphide in BPTI, and present evidence here that this is the case. A peptide model of [5-55], corresponding to a subdomain of BPTI, seems to fold into a native-like conformation, explaining why [5-55] does not lead to native protein and why it folds rapidly to N*. A native-like subdomain structure in a peptide model of [30-51], the other crucial one-disulphide intermediate, may explain the route by which [30-51] folds to native protein. Thus, much of the folding pathway of BPTI can be explained by the formation of a native-like subdomain in these two early intermediates. This suggests that a large part of the protein folding problem can be reduced to identifying and understanding subdomains of native proteins.

Previous work has shown that a synthetic peptide corresponding to the leucine zipper region of the yeast transcriptional activator GCN4 forms a stable dimer of alpha-helices and that the helices are oriented in a parallel manner. Two-dimensional nuclear magnetic resonance spectroscopy (NMR) is used here to demonstrate that the helix is continuous for at least 32 of the 33 residues in the peptide. The results also indicate that the dimer is symmetric. It is therefore unlikely that the interdigitation model for the structure of leucine zippers is correct, since interdigitation of leucine residues in a parallel dimer would lead to an asymmetric structure. The data are consistent with a coiled-coil structure.

Antibodies made to short peptides or to unfolded forms of proteins are often found to cross-react with intact proteins. These cross-reactions can be used to populate non-native protein conformations, possibly including protein folding intermediates, and the structures of the non-native conformations can be characterized using amide proton exchange and two-dimensional NMR.

A method for detecting structure in marginally stable forms of a protein is described. The principle is to measure amide proton exchange rates in the absence and presence of varying concentrations of a denaturant. Unfolding of structure by the denaturant is reflected by an acceleration of amide proton exchange rates, after correction for the effects of the denaturant on the intrinsic rate of exchange. This exchange-rate test for structure makes no assumptions about the rate of exchange in the unfolded state. The effects of 0-8 M urea and 0-6 M guanidinium chloride (GdmCl) on acid- and base-catalyzed exchange from model compounds have been calibrated. GdmCl does not appear to be well-suited for use in the exchange-rate test; model compound studies show that the effects of GdmCl on intrinsic exchange rates are complicated. In contrast, the effects of urea are a more uniform function of denaturant concentration. Urea increases acid-catalyzed, and decreases base-catalyzed, rates in model compounds. The exchange-rate test is used here to study structure formation in the S-protein (residues 21-124 of ribonuclease A). In conditions where an equilibrium folding intermediate of S-protein (I3) is known to be populated (pH 1.7, 0 degree C), the exchange-rate test for structure is positive. At higher temperatures (greater than 32 degrees C) I3 is unfolded, but circular dichroism data suggest that residual structure remains [Labhardt, A. M. (1982) J. Mol. Biol. 157, 357-371].(ABSTRACT TRUNCATED AT 250 WORDS)

In the folding reaction of the slow-folding species (US) of ribonuclease A (RNase A), the slow isomerization of wrong proline isomers provides a suitable trap for kinetic folding intermediates at low temperatures (0--10 degrees C). Partly folded intermediates are known to accumulate before proline isomerization takes place, after which native RNase A is formed. We have been able to measure the protection from amide proton exchange which is provided by structure in the intermediates at different times along the folding pathway. Previous work has shown that, by labeling the amide protons of the unfolded protein before initiating refolding, an early folding intermediate can be detected. The new pulse-labeling method presented here can be used to label later folding intermediates. Our results indicate that, in conditions which strongly favor the native protein, intermediates are formed which provide protection against exchange. However, when folding is initiated in 2.5 M Gdn . HCl, 10 degrees C, pH 7.5, conditions in which folding goes to completion but there are no spectroscopically detectable intermediates, then no intermediates are detected by our method. Alternate minimal mechanisms for the folding of US are presented.

Thioredoxin contains a single disulfide bond that can be reduced without perturbing significantly the structure of the enzyme. Upon reduction of the disulfide, protein stability decreases. We have experimentally tested the expected linkage relationship between disulfide bond formation and protein stability for thioredoxin. In order to do this, it is necessary to measure the equilibrium constant for disulfide bond formation in both the folded and unfolded states of the protein. Using glutathione as a reference species, we have measured the equilibrium constant for forming the disulfide bond (effective concentration) in thioredoxin as a function of urea concentration. As a control, we show that urea per se does not interfere with our measurements of thiol-disulfide equilibrium constants. Comparison of the values obtained for disulfide bond formation in the folded and unfolded states with the free energies for unfolding oxidized and reduced thioredoxin using circular dichroism confirms the expected linkage relationship. The urea dependence of thiol-disulfide equilibria provides a sensitive assay for folded structure in peptides or proteins. The method should also be useful to evaluate the stabilizing or destabilizing effect of natural or genetically engineered disulfides in proteins. In future work, the effects of amino acid substitutions on disulfide bond formation could be evaluated individually in the native and unfolded states of a protein.

The inactivation (desensitization) of the acetylcholine receptor by carbamylcholine, a stable analogue of acetylcholine, has been investigated in eel Ringer's solution, pH 7.0, 0 degrees C, by measurements of (i) ion flux and (ii) the kinetics of the reaction of [125I]-alpha-bungarotoxin with the receptor. The effect of preincubation with carbamylcholine is significantly different in the two types of measurement. In both the receptor-controlled flux of inorganic ions and the toxin-binding kinetics a biphasic process has been observed (Hess, G.P., Lipkowitz, S., Struve, G.E., 1978, Proc. Nat. Acad. Sci. USA 75:1703; Hess, G.P. et al., 1975, Biochem. Biophys. Res. Commun. 64:1018; Bulger, J.E. et al., 1977, Biochemistry 16:684), only the initial fast phase of which is inhibited and the subsequent slow phase persists. However, preincubation with carbamylcholine per se has no effect on the toxin reaction. The results obtained are consistent with the proposal of Katz and Thesleff (Katz, B., Thesleff, S., 1957, J. Physiol. (London) 138:65) that the active form of the receptor is converted to an inactive form in the presence of acetylcholine receptor ligands, and with our previous experiments (Hess et al., 1978) which indicated that one receptor form is responsible for the initial fast phase of both the receptor-controlled ion flux and the toxin binding reaction, and that its conversion to the other form results in the slow phases in these two measurements.

We present a case of soft-tissue reconstruction of the shoulder and upper arm utilizing a pedicled parascapular flap and discuss its potential as a flap for regional reconstruction.

A short peptide corresponding to the alpha-helical region of BPTI shows partial folding in aqueous solution (pH 7) as judged by circular dichroism (CD). Folding is temperature and denaturant sensitive, and the peptide is monomeric. The difference CD spectrum, obtained from spectra at two temperatures, indicates that the peptide folds as an alpha-helix. Difference CD spectroscopy provides a sensitive assay for helix formation in peptides exhibiting small amounts of structure. Helix stability in this peptide shows a marked pH dependence which is consistent with stabilizing charged side-chain interactions with the helix dipole and/or salt bridge formation.

Charged groups play a critical role in the stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A) in aqueous solution. One charged-group effect may arise from interactions between charged residues at either end of the helix and the helix dipole. We report here that studies of C-peptide analogues support the helix dipole model, and provide further evidence for the importance of electrostatic interactions not included in the Zimm-Bragg model for alpha-helix formation.

C-peptide, which contains the 13 NH2-terminal residues of RNase A, shows partial helix formation in water at low temperature (1 degree C, pH 5, 0.1 M NaCl), as judged by CD spectra; the helix is formed intramolecularly [Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476]. We find that helix stability depends strongly on pH: both a protonated histidine (residue 12) and a deprotonated glutamate (residue 9 or 2 or both) are required for optimal stability. This information, together with model building, suggests that the salt bridge Glu-9- ... His-12+ stabilizes the helix. Formation of the helix is enthalpy driven [van't Hoff delta H, - 16Kcal/mol (1 cal = 4.18 J)] and the helix is not observed above 30 degrees C. Proton NMR data indicate that several side chains adopt specific conformations as the helix is formed. These results have two implications for the mechanism of protein folding. First, they indicate that short alpha-helices, stabilized by specific side-chain interactions within the helix, can be stable enough in water to function as folding intermediates. Second, they suggest that similar experiments with peptides of controlled amino acid sequence could be used to catalogue the intrahelix interactions that stabilize or destabilize alpha-helices in aqueous solution. These data might provide the code relating amino acid sequence to the locations of alpha-helices in proteins.

We injected rabbits and guinea pigs with bovine thyrotropin (TSH) daily for 3 days, while controls received saline. All animals received sodium [125I]iodide on the second day, and thyroglobulin was purified from the thyroids of each group by gel filtration. Hormonogenic tryptic peptides from each S-cyanoethylated thyroglobulin preparation were isolated by high performance liquid chromatography, and their amino acid sequences were determined, permitting their localization within the thyroglobulin polypeptide chain by comparison with cDNA-derived sequences from bovine and human thyroglobulins. Thyroglobulins from the saline-injected rabbits and guinea pigs contained the same four major hormonogenic sites, designated A-D, previously described (Dunn, J. T., Anderson, P. C., Fox, J. W., Fassler, C. A., Dunn, A. D., Hite, L. A., and Moore, R. C. (1987) J. Biol. Chem. 262, 16948-16952). In both species, sites A and C were the major loci for thyroxine and triiodothyronine, respectively. However, site D in the guinea pig had a greater ratio of [125I]thyroxine to [127I]thyroxine than did site A, whereas the reverse was true in the rabbit. TSH administration produced the following changes in thyroglobulins of both species, relative to controls: 1) an increase in the ratio of [125I]triiodothyronine to [125I] thyroxine (rabbit, 0.29 versus 0.17; guinea pig, 0.19 versus 0.08), with the increase in triiodothyronine principally at site C; 2) a marked increase in 125I/127I and in thyroxine formation at site D (14.1% of thyroglobulin's thyroxine versus 9.8% in rabbits, 24 versus 13% in guinea pigs); 3) a corresponding decrease in thyroxine formation at site A (33 versus 43% in rabbits, 30 versus 46% in guinea pigs); and 4) a sharp increase in conversion of thyroglobulin's N-terminal 125I-labeled approximately 20 kDa hormone-rich iodopeptide, which contains site A, to a 125I-labeled approximately 15-kDa (rabbit) or 125I-labeled approximately 13-kDa (guinea pig) form, reflecting probable peptide bond cleavage. Our results show that TSH alters both the structure of the thyroglobulin molecule and the priority of utilization of its hormonogenic sites. We conclude that these changes are important to TSH's enhancement of thyroid hormone synthesis.