Increased folding stability of TEM-1 beta-lactamase by in vitro selection

In vitro selections of stabilized proteins lead to more robust enzymes and, at the same time, yield novel insights into the principles of protein stability. We employed Proside, a method of in vitro selection, to find stabilized variants of TEM-1 β-lactamase from Escherichia coli. Proside links the increased protease resistance of stabilized proteins to the infectivity of a filamentous phage. Several libraries of TEM-1 β-lactamase variants were generated by error-prone PCR, and variants with increased protease resistance were obtained by raising temperature or guanidinium chloride concentration during proteolytic selections. Despite the small size of phage libraries, several strongly stabilizing mutations could be obtained, and a manual combination of the best shifted the profiles for thermal unfolding and temperature-dependent inactivation of β-lactamase by almost 20 °C to a higher temperature. The wild-type protein unfolds in two stages: from the native state via an intermediate of the molten-globule type to the unfolded form. In the course of the selections, the native protein was stabilized by 27 kJ mol^− 1 relative to the intermediate and the cooperativity of unfolding was strongly increased. Three of our stabilizing replacements (M182T, A224V, and R275L) had been identified independently in naturally occurring β-lactamase variants with extended substrate spectrum. In these variants, they acted as global suppressors of destabilizations caused by the mutations in the active site. The comparison between the crystal structure of our best variant and the crystal structure of the wild-type protein indicates that most of the selected mutations optimize helices and their packing. The stabilization by the E147G substitution is remarkable. It removes steric strain that originates from an overly tight packing of two helices in the wild-type protein. Such unfavorable van der Waals repulsions are not easily identified in crystal structures or by computational approaches, but they strongly reduce the conformational stability of a protein.     

Stability of proteins in the presence of polyols estimated from their guanidinium chloride-induced transition curves at different pH values and 25 degrees C

We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.     

In vitro evolution of a hyperstable Gbeta1 variant

An in-vitro selection strategy was used to obtain strongly stabilized variants of the beta1 domain of protein G (Gbeta1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in Gbeta1 libraries that were created by error-prone PCR, and then, after randomization of these positions by saturation mutagenesis, strongly stabilized variants were selected. For both steps the in-vitro selection method Proside was employed. Proside links the stability of a protein with the infectivity of a filamentous phage. Ultimately, residues from the two best selected variants were combined in a single Gbeta1 molecule. This variant with the four mutations E15V, T16L, T18I, and N37L showed an increase of 35.1 degrees C in the transition midpoint and of 28.5 kJ mol(-1) (at 70 degrees C) in the Gibbs free energy of stabilization. It was considerably more stable than the best variant from a previous Proside selection, in which positions were randomized that had originally been identified by computational design. Only a single substitution (T18I) was found in both selections. The best variants from the present selection showed a higher cooperativity of thermal unfolding, as indicated by an increase in the enthalpy of unfolding by about 60 kJ mol(-1). This increase is apparently correlated with the presence of Leu residues that were selected at the positions 16 and 37.     

Relative Domain Folding and Stability of a Membrane Transport Protein

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately + 2 kcal mol^− 1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of + 2 kcal mol^− 1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains.     

Kinetic isotope effects reveal the presence of significant secondary structure in the transition state for the folding of the N-terminal domain of L9

Our present understanding of the nature of the transition state for protein folding depends predominantly on studies where individual side-chain contributions are mapped out by mutational analysis (phi value analysis). This approach, although extremely powerful, does not in general provide direct information about the formation of backbone hydrogen bonds. Here, we report the results of amide H/D isotope effect studies that probe the development of hydrogen bonded interactions in the transition state for the folding of a small alpha-beta protein, the N-terminal domain of L9. Replacement of amide protons by deuterons in a solvent of constant isotopic composition destabilized the domain, decreasing both its T(m) and Delta G(0) of unfolding. The folding rate also decreased. The parameter Phi(H/D), defined as the ratio of the effect of isotopic substitution upon the activation free energy to the equilibrium free energy was determined to be 0.6 in a D(2)O background and 0.75 in a H(2)O background, indicating that significant intraprotein hydrogen bond interactions are developed in the transition state for the folding of NTL9. The value is in remarkably good agreement with more traditional measures of the position of the transition state, which report on the relative burial of surface area. The results provide a picture of a compact folding transition state containing significant secondary structure. Indirect analysis argues that the bulk of the kinetic isotope effect arises from the beta-sheet-rich region of the protein, and suggests that the development of intraprotein hydrogen bonds in this region plays a critical role in the folding of NTL9.     

Molecular Determinants of a Native-State Prolyl Isomerization

Prolyl cis/trans isomerizations determine the rates of many protein-folding reactions, and they can serve as molecular switches and timers. The energy required to shift the prolyl cis/trans equilibrium during these processes originates from conformational reactions that are linked structurally and energetically with prolyl isomerization. We used the N2 domain of the gene-3-protein of phage fd to elucidate how such an energetic linkage develops in the course of folding. The Asp160-Pro161 bond at the tip of a β hairpin of N2 is cis in the crystal structure, but in fact, it exists as a mixture of conformers in folded N2. During refolding, about 10 kJ mol^− 1 of conformational energy becomes available for a 75-fold shift of the cis/trans equilibrium constant at Pro161, from 7/93 in the unfolded to 90/10 in the folded form. We combined single- and double-mixing kinetic experiments with a mutational analysis to identify the structural origin of this proline shift energy and to elucidate the molecular path for the transfer of this energy to Pro161. It originates largely, if not entirely, from the two-stranded β sheet at the base of the Pro161 hairpin. The two strands improve their stabilizing interactions when Pro161 is cis, and this stabilization is propagated to Pro161, because the connector peptides between the β strands and Pro161 are native-like folded when Pro161 is cis. In the presence of a trans-Pro161, the connector peptides are locally unfolded, and thus, Pro161 is structurally and energetically uncoupled from the β sheet. Such interrelations between local folding and prolyl isomerization and the potential modulation by prolyl isomerases might also be used to break and reestablish slow communication pathways in proteins.     

Key role of coulombic interactions for the folding transition state of the cold shock protein

The cold shock protein CspB shows a five-stranded beta-sheet structure, and it folds rapidly via a native-like transition state. A previous Phi value analysis showed that most of the residues with Phi values close to one reside in strand beta1, and two of them, Lys5 and Lys7 are partially exposed charged residues. To elucidate how coulombic interactions of these two residues contribute to the energetic organisation of the folding transition state we performed comparative folding experiments in the presence of an ionic denaturant (guanidinium chloride) and a non-ionic denaturant (urea) and a double-mutant analysis. Lys5 contributes 6.6 kJ mol(-1) to the stability of the transition state, and half of it originates from screenable coulombic interactions. Lys7 contributes 5.3 kJ mol(-1), and 3.4 kJ mol(-1) of it are screened by salt. In the folded protein Lys7 interacts with Asp25, and the screenable coulombic interaction between these two residues is fully formed in the transition state. This suggests that long-range coulombic interactions such as those originating from Lys5 and Lys7 of CspB can be important for organizing and stabilizing native-like structure early in protein folding.     

Thermodynamic properties of BPTI variants with highly simplified amino acid sequences

We report the first detailed thermodynamic analysis of simplified proteins by differential scanning calorimetry (DSC). The experiments were carried out with five simplified BPTI variants, whose structures and activities have been reported, in which several residues not essential for specifying the tertiary structure were replaced by alanine. In most aspects, the thermodynamics of simplified proteins were very similar to, if not essentially identical with, those of natural proteins. In particular, they undergo a highly cooperative two-state thermal unfolding process with a large enthalpy change, which is a thermodynamic hallmark of the native state of natural globular proteins. Furthermore, the specific enthalpy and entropy changes upon unfolding at 110 degrees C were close to values invariably observed for small natural globular proteins (55 J g(-1) and ~16 J K(-1) g(-1), respectively). On the other hand, two simplified BPTI variants, BPTI-21 and BPTI-22 (containing 21 and 22 alanine residues), were enthalpically stabilized while entropically destabilized with respect to the reference BPTI-[5,55] molecule. This peculiar type of entropy-enthalpy compensation is in sharp contrast to the usual enthalpy destabilization/entropy stabilization observed in mutational studies of natural proteins. Overall, we conclude that a thermodynamic native state can be achieved by proteins encoded with extensively simplified sequences.     

Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val→Thr or Phe→Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Φ-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (> 2.5 kJ mol^− 1) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.     

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ?-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.     

Effect of monomeric and oligomeric sugar osmolytes on ΔGD, the Gibbs energy of stabilization of the protein at different pH values: Is the sum effect of monosaccharide individually additive in a mixture?

Thermal denaturation curves of ribonuclease-A were measured by monitoring changes in the far-UV circular dichroism (CD) spectra in the presence of different concentrations of six sugars (glucose, fructose, galactose, sucrose, raffinose and stachyose) and mixture of monosaccharide constituents of each oligosaccharide at various pH values in the range of 6.0-2.0. These measurements gave values of T(m) (midpoint of denaturation), DeltaH(m) (enthalpy change at T(m)), DeltaC(p) (constant-pressure heat capacity change) under a given solvent condition. Using these values of DeltaH(m), T(m) and DeltaC(p) in appropriate thermodynamic relations, thermodynamic parameters at 25 degrees C, namely, DeltaG(D)(o) (Gibbs energy change), DeltaH(D)(o) (enthalpy change), and DeltaS(D)(o) (entropy change) were determined at a given pH and concentration of each sugar (including its mixture of monosaccharide constituents). Our main conclusions are: (i) each sugar stabilizes the protein in terms of T(m) and DeltaG(D)(o), and this stabilization is under enthalpic control, (ii) the protein stabilization by the oligosaccharide is significantly less than that by the equimolar concentration of the constituent monosaccharides, and (iii) the stabilization by monosaccharides in a mixture is fully additive. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of protein in their native and denatured states are not perturbed on the addition of sugars.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Optimization of the gbeta1 domain by computational design and by in vitro evolution: structural and energetic basis of stabilization

Computational design and in vitro evolution are major strategies for stabilizing proteins. For the four critical positions 16, 18, 25, and 29 of the B domain of the streptococcal protein G (Gbeta1), they identified the same optimal residues at positions 16 and 25, but not at 18 and 29. Here we analyzed the energetic contributions of the residues from these two approaches by single and double mutant analyses and determined crystal structures for a variant from the calculation (I16/L18/E25/K29) and from the selection (I16/I18/E25/F29). The structural analysis explains the observed differences in stabilization. Residues 16, 18, and 29 line an invagination, which results from a packing defect between the helix and the beta-sheet of Gbeta1. In all stabilized variants, residues with larger side-chains occur at these positions and packing is improved. In the selected variant, packing is better optimized than in the computed variant. Such differences in side-chain packing strongly affect stability but are difficult to evaluate by computation.     

Energetic coupling between native-state prolyl isomerization and conformational protein folding

In folded proteins, prolyl peptide bonds are usually thought to be either trans or cis because only one of the isomers can be accommodated in the native folded protein. For the N-terminal domain of the gene-3 protein of the filamentous phage fd (N2 domain), Pro161 resides at the tip of a beta hairpin and was found to be cis in the crystal structure of this protein. Here we show that Pro161 exists in both the cis and the trans conformations in the folded form of the N2 domain. We investigated how conformational folding and prolyl isomerization are coupled in the unfolding and refolding of N2 domain. A combination of single-mixing and double-mixing unfolding and refolding experiments showed that, in unfolded N2 domain, 7% of the molecules contain a cis-Pro161 and 93% of the molecules contain a trans-Pro161. During refolding, the fraction of molecules with a cis-Pro161 increases to 85%. This implies that 10.3 kJ mol(-1) of the folding free energy was used to drive this 75-fold change in the Pro161 cis/trans equilibrium constant during folding. The stabilities of the forms with the cis and the trans isomers of Pro161 and their folding kinetics could be determined separately because their conformational folding is much faster than the prolyl isomerization reactions in the native and the unfolded proteins. The energetic coupling between conformational folding and Pro161 isomerization is already fully established in the transition state of folding, and the two isomeric forms are thus truly native forms. The folding kinetics are well described by a four-species box model, in which the N2 molecules with either isomer of Pro161 can fold to the native state and in which cis/trans isomerization occurs in both the unfolded and the folded proteins.     

Dimer Formation of a Stabilized Gβ1 Variant: A Structural and Energetic Analysis

In previous work, a strongly stabilized variant of the β1 domain of streptococcal protein G (Gβ1) was obtained by an in vitro selection method. This variant, termed Gβ1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gβ1 gene libraries were performed, and the crystal structure of Gβ1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gβ1 by contributions of between 1.6 and 6.0 kJ mol^− 1 (at 70 °C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gβ1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-Å crystal structure of Gβ1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gβ1-M2 molecules via six intermolecular hydrogen bonds between the two β strands 2 and 2′ and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120° rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gβ1 variants.     

NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication

Slipped frameshift intermediates can occur when DNA polymerase slows or stalls at sites of DNA lesions. However, this phenomenon is much less common when unmodified DNA is replicated. In order to study the effect of templating bases on the alignment of primer-templates, NMR structural investigation has been performed on primer-template oligonucleotide models which mimic the situation that dNTP has just been incorporated opposite template. NMR evidence reveals the occurrence of misalignment when dGTP is incorporated opposite template T with a downstream nucleotide C. Depending on the template sequence, further extension of the primer can lead to realignment.     

Insertion of a chaperone domain converts FKBP12 into a powerful catalyst of protein folding

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.     

Energetics of protein folding

The energetics of protein folding determine the 3D structure of a folded protein. Knowledge of the energetics is needed to predict the 3D structure from the amino acid sequence or to modify the structure by protein engineering. Recent developments are discussed: major factors are reviewed and auxiliary factors are discussed briefly. Major factors include the hydrophobic factor (burial of non-polar surface area) and van der Waals interactions together with peptide hydrogen bonds and peptide solvation. The long-standing model for the hydrophobic factor (free energy change proportional to buried non-polar surface area) is contrasted with the packing-desolvation model and the approximate nature of the proportionality between free energy and apolar surface area is discussed. Recent energetic studies of forming peptide hydrogen bonds (gas phase) are reviewed together with studies of peptide solvation in solution. Closer agreement is achieved between the 1995 values for protein unfolding enthalpies in vacuum given by Lazaridis-Archontis-Karplus and Makhatadze-Privalov when the solvation enthalpy of the peptide group is taken from electrostatic calculations. Auxiliary factors in folding energetics include salt bridges and side-chain hydrogen bonds, disulfide bridges, and propensities to form alpha-helices and beta-structure. Backbone conformational entropy is a major energetic factor which is discussed only briefly for lack of knowledge.