Masahiro Iwakura

Systematic circular permutation of an entire protein reveals essential folding elements

The importance of chain connectivity in determining protein function and stability can be examined by breaking the peptide backbone using a technique such as circular permutation. Cleavage at certain positions results in a complete loss of the ability of the protein to fold. When such cleavage sites occur sequentially in the primary structure, we call the region a ‘folding element’, a new concept that could assist in our understanding of the protein folding problem. The folding elements of dihydrofolate reductase have been assigned by conducting a systematic circular permutation analysis in which the peptide backbone was sequentially broken between every pair of residues in the protein. The positions of folding elements do not appear to correspond to secondary structure motifs, substrate or coenzyme binding sites, or accessible surface area. However, almost all of the amino acid residues known to be involved in early folding events are located within the folding elements.

Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis

The flagellar hook–basal body (HBB) complex of the Gram‐positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins. The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook. There were no outer (P and L) rings that are found in Gram‐negative bacteria. The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium. The hook protein had an apparent molecular mass of 29 kDa, and its N‐terminal sequence was identical to that of B. subtilis FlgG, which was previously reported as a rod protein. The sequence of the reported FlgG protein of B. subtilis is more closely related to that of FlgE (the hook protein) rather than FlgG (the rod protein) of S. typhimurium, in spite of the difference of the apparent molecular masses between the two hook proteins (29 kDa versus 42 kDa). The hook–basal body contained six major proteins (with apparent molecular masses of 82, 59, 35, 32, 29 and 20 kDa) and two minor proteins (23 kDa and 13 kDa), which consistently appeared from preparation to preparation. The N‐terminus of each of these proteins was sequenced. Comparison with protein databases revealed the following polypeptide–gene correspondences: 82 kDa, fliF; 59 kDa, flgK; 35 kDa, orfF; 32 kDa, yqhF; 23 kDa, orf3 of the flaA locus; 20 kDa, flgB and flgC; 13 kDa, not determined. The band at 20 kDa was a mixture of FlgB and FlgC, as revealed by two‐dimensional gel analysis. Characteristic features of B. subtilis HBB are discussed in comparison with those of S. typhimiurium.

Microsecond subdomain folding in dihydrofolate reductase.

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR--in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse--emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.

Circular permutation analysis as a method for distinction of functional elements in the M20 loop of Escherichia coli dihydrofolate reductase.

A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia colidihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1–4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate inE. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of α carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following α helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates.

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher kcat value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-Å resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-l-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.

Stabilization of Hyperactive Dihydrofolate Reductase by Cyanocysteine-mediated Backbone Cyclization

Stabilization of an enzyme while maintaining its activity has been a major challenge in protein chemistry. Although it is difficult to simultaneously improve stability and activity of a protein by amino acid substitutions due to the activity-stability trade-off, backbone cyclization by connecting the N and C termini with a linker is promising as a general method of stabilizing a protein without affecting its activity. Recently, we created a hyperactive, methionine- and cysteine-free mutant of dihydrofolate reductase from Escherichia coli, called ANLYF, by introducing seven amino acid substitutions, which, however, destabilized the protein. Here we show that ANLYF is stabilized without a loss of its high activity by a novel backbone cyclization method for unprotected proteins. The method is based on the in vitro cyanocysteine-mediated intramolecular ligation reaction, which can be conducted with relatively high efficiency by a simple procedure and under mild conditions. We also show that the reversibility of thermal denaturation is highly improved by the cyclization. Thus, activity and stability of the protein can be separately improved by amino acid substitutions and backbone cyclization, respectively. We suggest that the cyanocysteine-mediated cyclization method is complementary to the intein-mediated cyclization method in stabilizing a protein without affecting its activity.

A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.

A novel method for peptide block synthesis using unprotected peptides

An S-cyanocysteine-mediated α-carbon activation reaction was used for the block synthesis of unprotected peptides. The repeated reaction using the S-cyanocysteinyl peptides as building blocks made it possible to ligate several peptide segments in order to synthesize larger peptides with natural amide bonding. Segment ligation reaction of X-cyanocysteinyl peptide with unprotected peptide (R2-NH2) is usuful for peptide block synthesis. Download full-size image

Protein Oxidation During Long Storage: Identification of the Oxidation Sites in Dihydrofolate Reductase from Escherichia coli through LC–MS and Fragment Studies

An LC-MS study revealed some heterogeneity in terms of molecular mass of a cysteine-free mutant of dihydrofolate reductase (DHFR) after long storage of the highly purified protein as an ammonium sulfate precipitate, but not in the case of a cysteine- and methioneine-free mutant of DHFR. One-third of the cysteine-free DHFR sample stored for a long time, around 18 months, comprised molecular species with molecular masses increased by 16, 32 and 48 Da. A peptide mapping study revealed that at least one of the methionine residues at positions 1, 16 and 20 was oxidatively modified to a methione-sulfoxide residue, while those at positions 42 and 92 were essentially unaffected. Each of the oxidized species of the DHFR exhibiting different degrees or sites of oxidation was further purified to essentially homogeneity in terms of molecular mass from the stored sample, and its enzyme activity was determined. One oxidized DHFR showed higher activity than that of the non-oxidized enzyme, while the other four oxidized DHFRs showed less activity. This agrees with the observation that the enzyme activity of the stored sample, a mixture in terms of oxidation, was apparently the same as that of the non-oxidized enzyme. This suggests that the activity itself is not a proper measure for quality control of proteins.

Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli.

One of the necessary conditions for a protein to be foldable is the presence of a complete set of “folding elements” (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE‐assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE–FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non‐native helices. MD simulations also show the non‐native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE–FE interactions, are also one of the necessary conditions for a protein to be foldable. Proteins 2006.