Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent

Although the denaturation of proteins by low temperatures is a well-documented phenomenon, little is known about the molecular details of the process. In this study, the parameters describing the denaturation thermodynamics of residues 6-85 of the N-terminal domain of lambda repressor have been determined by fitting the three-dimensional thermal-urea denaturation surface obtained by circular dichroism. The shape of the surface shows cold denaturation at low temperatures and urea concentrations above 2 M, which allows accurate determination of the apparent heat capacity of denaturation (delta Cp). Denaturation curves based on aromatic 1H NMR spectra give identical denaturation curves, confirming purely twostate folding under all conditions studies. The denaturation surface can be fit with constant delta Cp and delta In KD/delta[urea] (KD is the equilibrium constant for denaturation), consistent with a thermodynamically invariant denatured state. In addition, the aromatic 1H NMR spectrum of the cold denatured state at 0 degree C in 3 M uea is essentially identical to the spectrum at 70 degree C in 3 M urea. These observations indicate that the structures of the cold and heat denatured states, in the presence of 3 M urea, are thermodynamically and conformationally equivalent.     

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.     

Titration properties and thermodynamics of the transition state for folding: comparison of two-state and multi-state folding pathways

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (Dphys or I). CI2 folds from a largely unstructured denatured state under physiological conditions via a transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (Dphys-->++) is very similar for both proteins.     

Real-time NMR investigations of triple-helix folding and collagen folding diseases

Folding of the collagen triple helix provides an opportunity to look at multichain molecular assembly. This triple helix also offers unique advantages for the study of folding because the process is very slow compared to globular proteins, and the kinetics of folding can be obtained in real time by NMR. Studies on triple-helical peptides illustrate the ability to observe kinetic folding intermediates directly and the ability to propose detailed mechanisms of folding through the use of real-time NMR methods. Defective collagen folding has been implicated in various connective tissue diseases and the capacity of NMR to look at the folding of specific sites provides a tool for obtaining information about altered folding mechanisms. Comparison of folding in peptides that model normal and diseased collagens could shed light on the molecular perturbation and the etiology of disease.     

NMR and protein folding: equilibrium and stopped-flow studies

NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen-deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F-substituted amino acids to follow changes in side-chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site-directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped-flow device incorporated into the NMR spectrometer.     

Reversible association of the equilibrium unfolding intermediate of lambda Cro repressor

An extended differentiated scanning calorimetry study of the wild-type Cro repressor and of its V55C mutant has revealed a significant concentration dependence of the melting profiles, even though the two polypeptide chains forming the active repressor molecule are covalently bound within the mutant. An analysis of the temperature dependencies of the partial molar heat capacity suggests that in both cases equilibrium unfolding occurs via a highly-populated intermediate state corresponding to polypeptide tetramers. The results of thermodynamic analysis are confirmed by direct glutaraldehyde cross-linking experiments. Judging by heat effects and circular dichroism data, this intermediate state regains about 50% of the ordered structure and melts co-operatively.     

Salt effects on protein-DNA interactions. The lambda cI repressor and EcoRI endonuclease

In this paper, finite-difference solutions to the nonlinear Poisson-Boltzmann (NLPB) equation are used to calculate the salt dependent contribution to the electrostatic DNA binding free energy for both the lambda cI repressor and the EcoRI endonuclease. For the protein-DNA systems studied, the NLPB method describes nonspecific univalent salt dependent effects on the binding free energy which are in excellent agreement with experimental results. In these systems, the contribution of the ion atmosphere to the binding free energy substantially destabilizes the protein-DNA complexes. The magnitude of this effect involves a macromolecular structure dependent redistribution of both cations and anions around the protein and the DNA which is dominated by long range electrostatic interactions. We find that the free energy associated with global ion redistribution upon binding is more important than changes associated with local protein-DNA interactions (ion-pairs) in determining salt effects. The NLPB model reveals how long range salt effects can play a significant role in the relative stability of protein-DNA complexes with different structures.     

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.     

Active monomeric and dimeric forms of Pseudomonas putida glyoxalase I: evidence for 3D domain swapping

3D domain swapping of proteins involves the interconversion of a monomer containing a single domain-domain interface and a 2-fold symmetrical dimer containing two equivalent intermolecular interfaces. Human glyoxalase I has the structure of a domain-swapped dimer [Cameron, A. D., Olin, B., Ridderstr?m, M., Mannervik, B., and Jones, T. A. (1997) EMBO J. 16, 3386-3395] but Pseudomonas putida glyoxalase I has been reported to be monomeric [Rhee, H.-I., Murata, K., and Kimura, A. (1986) Biochem. Biophys. Res. Commun. 141, 993-999]. We show here that recombinant P. putida glyoxalase I is an active dimer (kcat approximately 500 +/- 100 s-1; KM approximately 0.4 +/- 0.2 mM) with two zinc ions per dimer. The zinc is required for structure and function. However, treatment of the dimer with glutathione yields an active monomer (kcat approximately 115 +/- 40 s-1; KM approximately 1.4 +/- 0.4 mM) containing a single zinc ion. The monomer is metastable and slowly reverts to the active dimer in the absence of glutathione. Thus, glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms. It is unique among domain-swapped proteins in that the active site and an essential metal binding site are apparently disassembled and reassembled by the process of domain swapping. Furthermore, it is the only example to date in which 3D domain swapping can be regulated by a small organic ligand.     

Structure prediction: folding proteins by pattern recognition

Although we are still a long way from being able to predict the details of protein structure from the underlying chemistry, slow but steady progress is being made at modeling structural features by recognizing the patterns that connect sequence to structure.     

A variant of lambda repressor with an altered pattern of cooperative binding to DNA sites

The bacteriophage lambda repressor binds cooperatively to pairs of adjacent sites in the lambda chromosome, one repressor dimer binding to each site. The repressor's amino domain (that which mediates DNA binding) is connected to its carboxyl domain (that which mediates dimerization and the interaction between dimers) by a protease-sensitive linker region. We have generated a variant lambda repressor that lacks this linker region. We show that dimers of the variant protein are deficient in cooperative binding to sites at certain, but not all, distances. The linker region thus extends the range over which carboxyl domains of DNA-bound dimers can interact. In particular, the linker is required for cooperative binding to a pair of sites as found in the lambda chromosome, and thus is essential for the repressor's physiological function.     

Direct NMR measurement of folding kinetics of a trimeric peptide

Direct NMR measurements of the folding kinetics are performed on a collagen-like triple helical peptide. The triple helical peptide was designed to model a biologically important region of collagen and has the sequence (POG)3ITGARGLAG(POG)4. Triple helical peptides were synthesized with specifically labeled 15N amino acid residues in key positions, and the kinetics of folding of the individual residues were monitored directly by measuring the loss of monomer intensity and the increase in trimer intensity. The residues at the terminal ends and central region could be followed independently and quantitated directly. Residues located at the terminal ends have rates and kinetics of folding that are distinct from residues in the central region of the peptide. This allows the monitoring of different steps in the folding mechanism and the postulation of the existence of a kinetic intermediate. The NMR data are consistent with a mechanism of association/nucleation and propagation. Hereditary connective tissue diseases are associated with mutations that result in abnormal folding of collagen, and the NMR folding experiments on a collagen-like peptide provide a basis for characterizing the molecular defect in folding mutations.     

Kinetic evidence for an on-pathway intermediate in the folding of cytochrome c

An early folding event of cytochrome c populates a helix-containing intermediate (INC) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U <-->INC <--> N, or whether the I must first move back through the U and then fold to the N through some alternative path (INC <--> U <--> N). By means of a kinetic test, it is shown here that the cytochrome c I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I <--> U <--> N option.     

NMR solution structure of the lead-dependent ribozyme: evidence for dynamics in RNA catalysis

The function of social calls emitted by foraging bats has received little study. Here we use observations of free-ranging greater spear-nosed bats, Phyllostomus hastatus, and field playbacks to determine whether audible, broad-band 'screech' calls attract mates, warn conspecifics or influence access to food. Five lines of evidence suggest that screech calls enable adult females from the same roosting group to fly together from the day roost to feeding sites. (1) Seasonal differences in diet influenced the rate of screech calling recorded outside the cave roost, as well as how often bats departed together. Bats called more often and flew in larger groups when feeding on a concentrated resource, balsa, Ochroma lagopus, flowers, in winter than on more dispersed Cecropia peltata fruit in spring. (2) Observations of bats flying outside the cave, in flyways and at feeding sites indicated that screech calls occurred more often when bats flew in groups than alone. (3) Females from the same roosting group were netted at the same feeding site, sometimes simultaneously, several kilometres from the cave. (4) Calling colour-marked adult females outside the cave were joined by a female group member, both on initial departures and on second foraging trips, more often than non-calling bats. (5) Playbacks attracted conspecifics at roost and feeding sites. Screech calls appear to function as contact calls that recruit and coordinate foraging among group members. We postulate that females benefit from foraging with unrelated roost-mates because they can defend feeding sites more effectively. Copyright 1998 The Association for the Study of Animal Behaviour.     

Real-time NMR studies on folding of mutants of barnase and chymotrypsin inhibitor 2

Apolipoprotein E (apo E) epsilon4 is a risk factor for sporadic and late-onset familial Alzheimer's disease, but it is not well known whether the apo E is associated with spinocerebellar degeneration. We studied the frequency of apo E allele in 59 olivopontocerebellar atrophy (OPCA) patients, including 13 pathologically confirmed cases. The distribution of the apo E allele frequency did not differ between OPCA patients and controls. Apo E allele does not influence the development of OPCA.     

NMR studies of the Escherichia coli Trp repressor.trpRs operator complex

To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy. We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.     

Kinetic evidence for an obligatory intermediate in the folding of the membrane protein bacteriorhodopsin

A photodiode array in conjunction with a rapid stopped-flow mixing method, with a millisecond time resolution, is used here to study the refolding of the membrane protein bacteriorhodopsin from an apoprotein state with a native-like secondary structure in mixed phospholipid/detergent micelles. Refolding to the native state is initiated by the rapid mixing of all-trans-retinal and the apoprotein bacterioopsin in mixed micelles. A lag phase of several seconds is observed in the appearance of the native state, as monitored by the increase in absorbance of the native chromophore. This observation demonstrates unequivocally that an intermediate is obligatory in the formation of bacteriorhodopsin. It is further shown that this intermediate is spectroscopically distinct from free retinal (absorbance maximum approximately 380 nm) and bacteriorhodopsin (absorbance maximum approximately 560 nm) and absorbs maximally at 430 nm. Evidence for the decay of the 430 nm intermediate into bacteriorhodopsin via three distinct parallel pathways is also provided. Taken together, these findings are used to describe a model in which distinct populations of the apoprotein in mixed micelles appear to fold along separate pathways via their corresponding intermediates into the native state. How the results of this study provide new insights into the mechanisms of protein folding is discussed.