Folding thermodynamics of a model three-helix-bundle protein

The calculated folding thermodynamics of a simple off-lattice three-helix-bundle protein model under equilibrium conditions shows the experimentally observed protein transitions: a collapse transition, a disordered-to-ordered globule transition, a globule to native-state transition, and the transition from the active native state to a frozen inactive state. The cooperativity and physical origin of the various transitions are explored with a single “optimization” parameter and characterized with the Lindemann criterion for liquid versus solid-state dynamics. Below the folding temperature, the model has a simple free energy surface with a single basin near the native state; the surface is similar to that calculated from a simulation of the same three-helix-bundle protein with an all-atom representation [Boczko, E. M. & Brooks III, C. L. (1995) Science 269, 393–396].     

Conformational fluctuations in single DNA molecules

Measurement of fluorescent lifetimes of dye-tagged DNA molecules reveal the existence of different conformations. Conformational fluctuations observed by fluorescence correlation spectroscopy give rise to a relaxation behavior that is described by “stretched” exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomogeneous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethylrhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational fluctuations. Fluorescence fluctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational fluctuations typical for single molecules are similar to those seen in single ion channels in cell membranes.     

The complementary aggregation sites of fibrin investigated through examination of polymers of fibrinogen with fragment E

Fibrin polymerizes through the interaction of sites exposed by the thrombin-mediated cleavage of fibrinopeptides in the central E region of the protein and complementary sites near the ends of the molecules, open in the D regions of both fibrinogen and fibrin. A preparation of fragment E, containing the central domain and part of the coiled-coil regions of fibrin, was used in mixtures with fibrinogen in this electron microscopy study to investigate the formation of fibrillar structures. At short times, linearly ordered oligomers of fibrinogen were observed with an additional mass of E fragments at the end-to-end junctions. At later times, long flexible polymers made up of 30 or more fibrinogen and fragment E units, with a tendency for lateral aggregation and tangle formation, were seen. These single-stranded assemblies could be readily dissociated in dilute acetic acid into their fibrinogen and fragment E components. However, if the aggregates were treated with factor XIIIa so that all γ chains became ligated by N(γ-glutamyl)lysine linkages, the polymers could no longer be taken apart. Because the only γ chains in the preparation are present in the fibrinogen molecules interacting end-to-end, the findings show that the factor XIIIa-induced cross-linking of γ chains in the clotting of fibrinogen or fibrin must occur between molecules that are longitudinal (or end-to-end) rather than transverse (or half-staggered).     

Electric field-induced reorganization of two-component supported bilayer membranes

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory’s approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi–Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.     

Is the molten globule a third phase of proteins?

The equilibrium properties of proteins are studied by Monte Carlo simulation of two simplified models of protein-like heteropolymers. These models emphasize the polymeric entropy of the fluctuating polypeptide chain. Our calculations suggest a generic phase diagram that contains a thermodynamically distinct “molten globule” state in addition to a rigid native state and a nontrivial unfolded state. The roles of side-chain packing and loop entropy are discussed.     

Novel mechanism for carbamoyl-phosphate synthetase: A nucleotide switch for functionally equivalent domains

Carbamoyl-phosphate synthetases (CPSs) utilize two molecules of ATP at two internally duplicated domains, B and C. Domains B and C have recently been shown to be structurally [Thoden, J. B., Holden, H. M., Wesenberg, G., Raushel, F. M. & Rayment, I. (1997) Biochemistry 36, 6305–6316] and functionally [Guy, H. I. & Evans, D. R. (1996) J. Biol. Chem. 271, 13762–13769] equivalent. We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A/B motif in domains B and C. To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS. In this mechanism utilization of ATP bound to domain C is coupled to carbamoyl-phosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.     

Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein

The 2.1-Å resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 → Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore’s excited state to Glu-222. Loss of the Ser-205–Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.     

Monomolecular collapse of plasmid DNA into stable virus-like particles

Cationic lipids are being widely used for cell transfection in vitro. The lipid/DNA complexes, however, tend to aggregate into large and polydisperse particle mixtures; this hampers their use in vivo. Cationic detergents, on the contrary, do not mediate cell transfection per se, yet are capable of condensing individual DNA molecules into discrete entities. We have taken (only) the interesting features of both types of amphiphiles for the two-step formation of stable core particles reminiscent of viruses. Individual anionic plasmid molecules were cooperatively collapsed with a carefully tailored cationic cysteine-based detergent. The resulting 23-nm particles were then simply “frozen” by spontaneous aerobic dimerization of the cysteine-detergent into a cystine-lipid on the template DNA. The population of spherical particles is monodisperse and stable over days, in physiological conditions. Together with a negative surface potential, these properties should ensure good tissue dissemination and escape from the blood stream after i.v. injection.     

Two-state models of protein folding kinetics

The folding of some proteins appears to be a two-state kinetic process. A two-state kinetic model is justified if protein molecules rapidly equilibrate between different unfolded conformations prior to complete folding. Here I show that this rapid equilibration is a natural consequence of reasonable assumptions about reaction rate constants and folding thermodynamics.     

Probes bound to myosin Cys-707 rotate during length transients in contraction

It is widely conjectured that muscle shortens because portions of myosin molecules (the “cross-bridges”) impel the actin filament to which they transiently attach and that the impulses result from rotation of the cross-bridges. Crystallography indicates that a cross-bridge is articulated–consisting of a globular catalytic/actin-binding domain and a long lever arm that may rotate. Conveniently, a rhodamine probe with detectable attitude can be attached between the globular domain and the lever arm, enabling the observer to tell whether the anchoring region rotates. Well-established signature effects observed in shortening are tension changes resulting from the sudden release or quick stretch of active muscle fibers. In this investigation we found that closely correlated with such tension changes are changes in the attitude of the rhodamine probes. This correlation strongly supports the conjecture about how shortening is achieved.     

Activation of protein kinase C by tyrosine phosphorylation in response to H2O2

Protein kinase C (PKC) isoforms, α, βI, and γ of cPKC subgroup, δ and of nPKC subgroup, and ζ of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H₂O₂. These isoforms isolated from the H₂O₂-treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC α and δ, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC δ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H₂O₂. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.     

Membrane-promoted unfolding of acetylcholinesterase: A possible mechanism for insertion into the lipid bilayer

Acetylcholinesterase from Torpedo californica partially unfolds to a state with the physicochemical characteristics of a “molten globule” upon mild thermal denaturation or upon chemical modification of a single nonconserved buried cysteine residue, Cys²³¹. The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys²³¹ by mercuric chloride produces initially a quasi-native state of Torpedo acetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1–2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteins in situ.     

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La³⁺ concentration in the presence of a constant concentration of Mg²⁺. We show that a bell-shaped curve of cleavage activation is obtained as La³⁺ is added in micromolar concentrations in the presence of 8 mM Mg²⁺, with a maximal rate of cleavage being attained in the presence of 3 μM La³⁺. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the K_d values for Mg²⁺ of 3.5 mM and >50 mM, that these sites bind La³⁺ with estimated K_d values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg²⁺ by La³⁺ at the stronger (relative to Mg²⁺) binding site activating catalysis and displacement of Mg²⁺ by La³⁺ at the weaker (relative to Mg²⁺) (relative to Mg²⁺) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pK_a of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg²⁺–La³⁺ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.     

A heparin-binding synthetic peptide of heparin/heparan sulfateinteracting protein modulates blood coagulation activities

We have previously identified and characterized a heparin-binding cell surface protein (heparin/heparan sulfate-interacting protein, or HIP) present on epithelial and endothelial cells. A synthetic peptide mimicking a heparin-binding domain of HIP is now shown to bind a small subset of heparin molecules with high affinity and, therefore, presumably recognizes a specific structural motif in the heparin molecule. Further analyses revealed that the heparin molecules exhibiting a high affinity for the HIP peptide also show an extremely high affinity for antithrombin III (AT-III), a cofactor required for heparin’s anticoagulant activity. The HIP peptide was shown to compete with AT-III for binding to heparin and to neutralize the anticoagulant activity of heparin in blood plasma assays. Furthermore, the heparin subfraction that binds to the HIP peptide with high affinity exhibits an extremely high anticoagulant activity. We conclude that although the HIP peptide shows no sequence similarity with AT-III, the two proteins recognize the same or similar structural motifs in heparin.     

Proteins unfold in steps

I present results from an experiment on the dynamics of folding of a globular protein (bovine serum albumin). Employing a micro-mechanical technique, I perform the measurements on very few molecules (1–100). I observed a sequence of steps in time for both unfolding and refolding. The overall characteristic time of the process is thus built up of waiting times between successive steps. The pattern of steps is reproducible, demonstrating the existence of deterministic pathways for folding and unfolding. Certain symmetries in the patterns of steps may reflect the architecture of the protein’s structure.     

AlF3 mimics the transition state of protein phosphorylation in the crystal structure of nucleoside diphosphate kinase and MgADP

Nucleoside diphosphate kinase reversibly transfers the γ-phosphate of ATP onto its active site histidine. We have investigated the transition state of histidine phosphorylation with the high-resolution crystal structures of the enzyme from Dictyostelium discoideum with MgADP and either aluminium or beryllium fluoride. The bound aluminium fluoride species is the neutral species AlF₃ and not the more common AlF₄⁻. AlF₃ forms a trigonal bipyramid that makes it an accurate analog of the transition state of the γ-phosphate of ATP undergoing transfer to the catalytic histidine. Its axial ligands are a histidine nitrogen and a β-phosphate oxygen. Beryllium fluoride also binds at the same position and with the same ligands but in a tetrahedral geometry resembling the Michaelis complex rather than the transition state. The two x-ray structures show explicit enzyme–substrate interactions that discriminate between the ground and the transition states of the reaction. They also illustrate the partially dissociative geometry of the transition state of phosphoryl transfer and demonstrate the potential applications of metallofluorides for the study of kinase mechanisms.     

Phosphatidylinositol 3-kinase-γ activates Bruton’s tyrosine kinase in concert with Src family kinases

Bruton’s tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bind in vitro to several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-γ. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-γ and Src family kinases.     

Chiral molecular self-assembly of phospholipid tubules: A circular dichroism study

We report on spectroscopic studies of the chiral structure in phospholipid tubules formed in mixtures of alcohol and water. Synthetic phospholipids containing diacetylenic moieties in the acyl chains self-assemble into hollow, cylindrical tubules in appropriate conditions. Circular dichroism provides a direct measure of chirality of the molecular structure. We find that the CD spectra of tubules formed in mixtures of alcohol and water depends strongly on the alcohol used and the lipid concentration. The relative spectral intensity of different circular dichroism bands correlates with the number of bilayers observed using microscopy. The results provide experimental evidence that tubule formation is based on chiral packing of the lipid molecules and that interbilayer interactions are important to the tubule structure.     

Computer-based analysis of the binding steps in protein complex formation

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components—an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple “ball-and-stick” structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and K_ds and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.     

Structural correlations in protein folding funnels

While the overall energy landscape of a foldable protein can be described by means of a few parameters characterizing its statistical topography, specific energetic terms subtly bias the representative structures giving rise to residue pair correlations as in a liquid. We use a free energy functional incorporating an inhomogeneous pair contact energy along with a contact formation entropy and a cooperativity contribution to determine residue-specific contact probabilities in the denatured state and the transition state ensemble. The predicted “hot residues” for the theoretical transition state ensemble reasonably agree with experiment for chymotrypsin inhibitor 2, and generally a strong correlation exists with the measured kinetic effects of mutating residues not involved in highly solvent-exposed regions.