Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli

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.     

The effects of Escherichia coli sepsis and short-term ischemia on coronary vascular reactivity and myocardial function

A moderately stable protein with typical folding kinetics unfolds and refolds many times during its cellular lifetime. In monomeric lambda repressor this process is extremely rapid, with an average folded state lifetime of only 30 milliseconds. A thermostable variant of this protein (G46A/G48A) unfolds with the wild-type rate, but it folds in approximately 20 microseconds making it the fastest-folding protein yet observed. The effects of alanine to glycine substitutions on the folding and unfolding rate constants of the G46A/G48A variant, measured by dynamic NMR spectroscopy, indicate that the transition state is an ensemble comprised of a disperse range of conformations. This structural diversity in the transition state is consistent with the idea that folding chains are directed towards the native state by a smooth funnel-like conformational energy landscape. The kinetic data for the folding of monomeric lambda repressor can be understood by merging the new energy landscape view of folding with traditional models. This hybrid model incorporates the conformational diversity of denatured and transition state ensembles, a transition state activation energy, and the importance of intrinsic helical stabilities.     

Thermodynamic analysis of alpha-spectrin SH3 and two of its circular permutants with different loop lengths: discerning the reasons for rapid folding in proteins

The temperature dependences of the unfolding-refolding reaction of a shorter version of the alpha-spectrin SH3 domain (PWT) used as a reference and of two circular permutants (with different poly-Gly loop lengths at the newly created fused loop) have been measured by differential scanning microcalorimetry and stopped-flow kinetics, to characterize the thermodynamic nature of the transition and native states. Differential scanning calorimetry results show that all these species do not belong to the same temperature dependency of heat effect. The family of the N47-D48s circular permutant (with 0-6 Gly inserted at the fused-loop) shows a higher enthalpy as happens with the PWT domain. The wild type (WT) and the S19-P20s permutant family have a more similar behavior although the second is far less stable. The crystallographic structure of the PWT shows a hairpin formation in the region corresponding to the unstructured N-terminus tail of the WT, explaining the enthalpic difference. There is a very good correlation between the calorimetric changes and the structural differences between the WT, PWT, and two circular permutants that suggests that their unfolded state cannot be too different. Elongation of the fused loop in the two permutants, taking as a reference the protein with one inserted Gly, results in a small Gibbs energy change of entropic origin as theoretically expected. Eyring plots of the unfolding and refolding semireactions show different behaviors for PWT, S19-P20s, and N47-D48s in agreement with previous studies indicating that they have different transition states. The SH3 transition state is relatively close to the native state with regard to changes in heat capacity and entropy, indicating a high degree of compactness and order. Regarding the differences in thermodynamic parameters, it seems that rapid folding could be achieved in proteins by decreasing the entropic barrier.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.     

p53 protein accumulation and response to adjuvant chemotherapy in premenopausal women with node-negative early breast cancer

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Dynamics of the conformational ensemble of partially folded bovine pancreatic trypsin inhibitor

A single-disulfide variant of bovine pancreatic trypsin inhibitor (BPTI), [14-38]Abu, is a partially folded ensemble which includes two, and in one case three, conformations that interconvert slowly enough to exhibit separate cross-peaks in the amide region of homonuclear and heteronuclear NMR spectra. Each conformation is itself composed of many subconformations in rapid equilibrium. Partially folded BPTI undergoes local motions that are slow, noncooperative, independent fluctuations of short segments within the chain. Cooperative global unfolding of the ensemble is also observed. Heteronuclear NMR has been used to measure interconversion rate constants of partially folded conformational substates; the rate constants differ for each residue and vary over an order of magnitude. For local fluctuation, the forward rate constants for amide protons of the antiparallel beta-sheet are significantly smaller than the rest of the molecule, consistent with other indications that this is the most stable part of the partially folded protein. The reverse rate constants also vary; they are the highest for Ala 27 in the turn between the strands in the sheet and for Phe 33 in the antiparallel beta-sheet. Global unfolding interconversion rate constants vary over a 3-fold range, consistent with previously observed deviations from two-state behavior. Fast backbone dynamics, from T1, T2, and NOE relaxation parameters, are obtained for the slowly interchanging conformations in the partially folded ensemble. Clear differences are observed between the two conformations; one is more flexible and less compact than the other. In the more flexible and disordered partially folded conformation, intermediate exchange is detected for some backbone amides, namely, those in the central beta-sheet and the turn. These same sheet and turn residues are more ordered in the globally denatured ensemble as well. Our results suggest that the turn initiates formation of a partially folded ensemble in which the slow-exchange core is the most stable region and in which segmental fluctuations reflect multiple nuclei for folding of the rest of the molecule.     

Non-native local interactions in protein folding and stability: introducing a helical tendency in the all beta-sheet alpha-spectrin SH3 domain

The relative importance of secondary structure interactions versus tertiary interactions for stabilising and guiding the folding process is a matter for discussion. Phenomenological models of protein folding assign an important role to local contacts in protein folding and stability. On the other hand, simplistic lattice simulations find that secondary structure is mainly the product of protein compaction and that optimisation of folding speed seems to require small contributions of local contacts to the stability of the folded state. To examine the extent to which secondary structure propensities influence protein folding and stability, we have designed mutations that introduce a strong non-native helical propensity in the first 19 residues of the alpha-spectrin SH3 domain. The mutant proteins have the same three-dimensional structure as the wild-type, but they are less stable and have less co-operative folding transitions. There seems to be a relationship between the non-native helical propensity and the compaction of the denatured state. This suggests that in the denatured ensemble under native conditions there is a significant proportion of compact structures with non-native secondary structures. Our results demonstrate that non-local interactions can overcome strong non-native secondary structure propensities and, more important, that optimisation of folding speed and co-operativity requires the latter to be relatively small.     

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.     

Crystal structure of the IIB subunit of a fructose permease (IIBLev) from Bacillus subtilis

The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates both the uptake of carbohydrates across the cytoplasmic membrane and their phosphorylation. During this process, a phosphoryl group is transferred from phosphoenolpyruvate via the general PTS proteins enzyme I, HPr and the sugar-specific components IIA, IIB to the transported sugar. The crystal structure of the IIB subunit of a fructose transporter from Bacillus subtilis (IIBLev) was solved by MIRAS to a resolution of 2.9 A. IIBLev comprises 163 amino acid residues that are folded into an open, mainly parallel beta-sheet with helices packed on either face. The phosphorylation site (His15) is located on the first loop (1/A) at one of the topological switch-points of the fold. Despite different global folds, IIBLev and HPr have very similar active-site loop conformations with the active-site histidine residues located close to the N terminus of the first helix. This resemblance may be of functional importance, since both proteins exchange a phosphoryl group with the same IIA subunit. The structural basis of phosphoryl transfer from HPr to IIAMan to IIBMan was investigated by modeling of the respective transition state complexes using the known HPr and IIAMan structures and a homology model of IIBMan that was derived from the IIBLev structure. All three proteins contain a helix that appears to be suitable for stabilization of the phospho-histidine by dipole and H-bonding interactions. Smooth phosphoryl transfer from one N-cap position to the other appears feasible with a minimized transition state energy due to simultaneous interactions with the donor and the acceptor helix.     

High populations of non-native structures in the denatured state are compatible with the formation of the native folded state

The structures of the denatured states of the spectrin SH3 domain and a mutant designed to have a non-native helical tendency at the N terminus have been analyzed under mild acidic denaturing conditions by nuclear magnetic resonance methods with improved resolution. The wild-type denatured state has little residual structure. However, the denatured state of the mutant has an approximately 50% populated helical structure from residues 2 to 14, a region that forms part of the beta-sheet structure in the folded state. Comparison with a peptide corresponding to the same sequence shows that the helix is stabilized in the whole domain, likely by non-local interactions with other parts of the protein as suggested by changes in a region far from the mutated sequence. These results demonstrate that high populations of non-native secondary structure elements in the denatured state are compatible with the formation of the native folded structure.     

Serum sensitivity and cell surface hydrophobicity of Klebsiella pneumoniae treated with gentamicin, tobramycin and amikacin

The individual chains in the triple helix of collagen occur in a conformation related to polyproline II because of the presence of large number of imino peptide bonds. However, these residues are not evenly distributed in the collagen molecule which also contains many non-imino residues. These non-imino regions of collagen may be expected to show preference for other than triple helical conformations. The appearance of several Raman bands in solution phase at 65 degrees C raises the possibility of non-uniform triple helical structure in collagen. Raman spectroscopic studies on collagen in the solid state and in solution at a temperature greater than its denaturation temperature, reported here suggest that denatured collagen may exhibit an ensemble of conformational states with yet unknown implications to the biochemical interactions of this important protein component of connective tissues.     

The changing nature of the protein folding transition state: implications for the shape of the free-energy profile for folding

According to landscape theory proteins do not fold by localised pathways, but find their native conformation by a progressive organisation of an ensemble of partly folded structures down a folding funnel. Here, we use kinetics and protein engineering to investigate the shape of the free-energy profile for two-state folding, which is the macroscopic view of the funnel process for small and rapidly folding proteins. Our experiments are based mainly on structural changes of the transition state of chymotrypsin inhibitor 2 (CI2) upon destabilisation with temperature and GdnHCl. The transition state ensemble of CI2 is a localised feature in the free-energy profile that is sharply higher than the other parts of the activation barrier. The relatively fixed position of the CI2 transition state on the reaction coordinate makes it easy to characterise but contributes also to overshadow the rest of the free-energy profile, the shape of which is inaccessible for analysis. Results from mutants of CI2 and comparison with other two-state proteins, however, point at the possibility that the barrier for folding is generally broad and that localised transition states result from minor ripples in the free-energy profile. Accordingly, variabilities in the folding kinetics may not indicate different folding mechanisms, but could be accounted for by various degrees of ruggedness on top of very broad activation barriers for folding. The concept is attractive since it summarises a wide range of folding data which have previously seemed unrelated. It is also supported by theory. Consistent with experiment, broad barriers predict that new transition state ensembles are exposed upon extreme destabilisation or radical mutations.     

Osmolyte-driven contraction of a random coil protein

The Stokes radius characteristics of reduced and carboxamidated ribonuclease A (RCAM RNase) were determined for transfer of this "random coil" protein from water to 1 M concentrations of the naturally occurring protecting osmolytes trimethylamine N-oxide, sarcosine, sucrose, and proline and the nonprotecting osmolyte urea. The denatured ensemble of RCAM RNase expands in urea and contracts in protecting osmolytes to extents proportional to the transfer Gibbs energy of the protein from water to osmolyte. This proportionality suggests that the sum of the transfer Gibbs energies of individual parts of the protein is responsible for the dimensional changes in the denatured ensemble. The dominant term in the transfer Gibbs energy of RCAM RNase from water to protecting osmolytes is the unfavorable interaction of the osmolyte with the peptide backbone, whereas the favorable interaction of urea with the backbone dominates in RCAM RNase transfer to urea. The side chains collectively favor transfer to the osmolytes, with some protecting osmolytes solubilizing hydrophobic side chains as well as urea does, a result suggesting there is nothing special about the ability of urea to solubilize hydrophobic groups. Protecting osmolytes stabilize proteins by raising the chemical potential of the denatured ensemble, and the uniform thermodynamic force acting on the peptide backbone causes the collateral effect of contracting the denatured ensemble. The contraction decreases the conformational entropy of the denatured state while increasing the density of hydrophobic groups, two effects that also contribute to the ability of protecting osmolytes to force proteins to fold.     

Dependence of protein stability on the structure of the denatured state: free energy calculations of I56V mutation in human lysozyme

Free energy calculations were carried out to understand the effect of the I56V mutation of human lysozyme on its thermal stability. In the simulation of the denatured state, a short peptide including the mutation site in the middle is employed. To study the dependence of the stability on the denatured-state structure, five different initial conformations, native-like, extended, and three random-coil-like conformations, were examined. We found that the calculated free energy difference, DeltaDeltaGcal, depends significantly on the structure of the denatured state. When native-like structure is employed, DeltaDeltaGcal is in good agreement with the experimental free energy difference, DeltaDeltaGexp, whereas in the other four models, DeltaDeltaGcal differs sharply from DeltaDeltaGexp. It is therefore strongly suggested that the structure around the mutation site takes a native-like conformation rather than an extended or random-coil conformation. From the free energy component analysis, it has been shown that free energy components originating from Lennard-Jones and covalent interactions dominantly determine DeltaDeltaGcal. The contribution of protein-protein interactions to the nonbonded component of DeltaDeltaGcal is about the same as that from protein-water interactions. The residues that are located in a hydrophobic core (F3, L8, Y38, N39, T40, and I89) contribute significantly to the nonbonded free energy component of DeltaDeltaGcal. We also propose a general computational strategy for the study of protein stability that is equally conscious of the denatured and native states.     

Metal coordination of azurin in the unfolded state

1H NMR data applied to the paramagnetic cobalt(II) derivative of azurin from Pseudomonas aeruginosa have made it possible to show that the metal ion is bound to the protein in the unfolded state. The relaxation data as well as the low magnetic anisotropy of the metal ion indicate that the cobalt ion is tetrahedral in the unfolded form. The cobalt ligands have been identified as the residues Gly45, His46, Cys112 and His117. Met121 is not coordinated in the unfolded state. In this state, the metal ion is not constrained to adopt a bipyramidal geometry, as imposed by the protein when it is folded. This is clear confirmation of the rack-induced bonding mechanism previously proposed for the metal ion in azurin.     

Exploring the folding funnel of a polypeptide chain by biophysical studies on protein fragments

We are examining possible roles of native and non-native interactions in early events in protein folding by a systematic analysis of the structures of fragments of proteins whose folding pathways are well characterised. Seven fragments of the 110-residue protein barnase, corresponding to the progressive elongation from its N terminus, have been characterised by a battery of biophysical and spectroscopic methods. Barnase is a multi-modular protein that folds via an intermediate in which the C-terminal region of its major alpha-helix (alpha-helix1, residues Thr6-His18) is substantially formed as is also its anti-parallel beta-sheet, centred around a beta-hairpin (residues Ser92-Leu95). Fragments up to, and including, residues 1-95 (fragment B95), appeared to be mainly disordered, although a small amount of helical secondary structure in each was inferred from far-UV CD experiments, and fluorescence studies indicated some native-like tertiary interactions in B95. The largest fragment (residues 1-105, B105) is compactly folded. The secondary structure in alpha-helix1 in the seven fragments was found by NMR to increase with increasing chain length faster than the build-up of tertiary interactions, indicating that alpha-helix1 is being stabilised by non-native interactions. This behaviour contrasts with that in fragments of the 64-residue chymotrypsin inhibitor 2 (CI2), in which tertiary and secondary structures build up in parallel with increasing length. CI2 consists of a single module of structure that folds without a detectable intermediate. The largest fragment of barnase, B105, has interactions that resemble its folding intermediate, whereas one of the largest fragments of CI2 (residues 1-60) resembles the folding transition state. The folding pathways of both proteins are consistent with a scheme in which there are low levels of native-like secondary structure in the denatured state that become stabilised by long-range interactions as folding proceeds. Neither protein forms a stable fold when lacking the last ten residues at the C terminus. Since at least 20 amino acid residues are bound to the ribosome during protein biosynthesis, these small proteins do not fold until they have left the ribosome, and so the studies of the folding of such proteins in vitro may be relevant to their folding in vivo, especially as the molecular chaperone GroEL binds only weakly to denatured CI2 and does not discernibly alter the folding mechanism of barnase.     

Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate

We investigated the role of a partially folded intermediate that transiently accumulates during lysozyme folding. Previous studies had shown that the partially folded intermediate is located on a slow-folding pathway and that an additional fast direct pathway from the unfolded state to the native state exists. Kinetic double-jump experiments showed that the two folding pathways are not caused by slow equilibration reactions in the unfolded state. Rather, kinetic partitioning occurs very early in lysozyme refolding, giving the molecules the chance to enter the direct pathway or a slow-folding channel. Fitting the guanidinium chloride dependencies of the refolding and unfolding reactions to analytical solutions for different folding scenarios enables us to propose a triangular mechanism as the minimal model for lysozyme folding explaining all observed kinetic reactions: [diagram in text]. All microscopic rate constants and their guanidinium chloride dependencies could be obtained from the experimental data. The results suggest that population of the intermediate during refolding increases the free energy of activation of the folding process. This effect is due to the increased stability of the intermediate state compared to the unfolded state leading to an increase in the free energy of activation (deltaG0) compared to folding in the absence of populated intermediate states. The absolute energy of the transition state is identical on both pathways. The results imply that pre-formed secondary structure in the folding intermediate obstructs formation of the transition state of folding but does not change the nature of the rate-limiting step in the folding process.     

Satisfying turns in folding transitions

Protein engineering studies show that conformations in the folding transition state ensemble can be structurally polarized. In two SH3 beta-sheet domains, the formation of hydrophobic contacts goes hand in hand with the formation of the solvated distal loop beta-turn, while large parts of the molecule remain unstructured in the ensemble.     

Probing the structure and mechanism of Ras protein with an expanded genetic code

Mutations in Ras protein at positions Gly12 and Gly13 (phosphate-binding loop L1) and at positions Ala59, Gly60, and Gln61 (loop L4) are commonly associated with oncogenic activation. The structural and catalytic roles of these residues were probed with a series of unnatural amino acids that have unusual main chain conformations, hydrogen bonding abilities, and steric features. The properties of wild-type and transforming Ras proteins previously thought to be uniquely associated with the structure of a single amino acid at these positions were retained by mutants that contained a variety of unnatural amino acids. This expanded set of functional mutants provides new insight into the role of loop L4 residues in switch function and suggests that loop L1 may participate in the activation of Ras protein by effector molecules.     

Monitoring the sizes of denatured ensembles of staphylococcal nuclease proteins: implications regarding m values, intermediates, and thermodynamics

Fluorescence and size-exclusion chromatography (SEC) are used to monitor urea denaturation of wild-type staphylococcal nuclease (SN) as well as the m+ and m- mutants A69T and V66W, respectively. It is found that the SEC partition coefficient, 1/Kd, is directly proportional to the Stokes radii of proteins. From the Stokes radii, the denatured ensembles of the three proteins are found to be highly compact in the limit of low urea concentration and expand significantly with increasing urea concentration. The m values from fluorescence-detected denaturation of the SN proteins are generally considered to reflect the relative sizes of denatured ensembles. However, the rank order of m values of the SN proteins studied do not correspond to the rank order of denatured ensemble sizes detected by 1/Kd, suggesting that m values reflect more than just surface area increases on denaturation. SEC provides two complementary ways to demonstrate the existence of intermediates in urea denaturation and illustrates that V66W undergoes a three-state transition. Fluorescence-detected urea denaturations of A69T and wt SN do not correspond with 1/Kd-detected denaturation profiles, a result that would ordinarily mean that the transitions are non-two-state. However, this interpretation fails to recognize the rapidly changing size and thermodynamic character of the denatured ensembles of these proteins both within and outside of the transition zone. The implications of the changing sizes and thermodynamic character of the denatured ensembles for SN proteins are manifold, requiring a reconsideration of the thermodynamics of proteins whose denatured ensembles behave as those of SN proteins.