The building block folding model and the kinetics of protein folding.

Here we show that qualitatively, the building blocks folding model accounts for three-state versus the two-state protein folding. Additionally, it is consistent with the faster versus slower folding rates of the two-state proteins. Specifically, we illustrate that the building blocks size, their mode of associations in the native structure, the number of ways they can combinatorially assemble, their population times and the way they are split in the iterative, step-by-step structural dissection which yields the anatomy trees, explain a broad range of folding rates. We further show that proteins with similar general topologies may have different folding pathways, and hence different folding rates. On the other hand, the effect of mutations resembles that of changes in conditions, shifting the population times and hence the energy landscapes. Hence, together with the secondary structure type and the extent of local versus non-local interactions, a coherent, consistent rationale for folding kinetics can be outlined, in agreement with experimental results. Given the native structure of a protein, these guidelines enable a qualitative prediction of the folding kinetics. We further describe these in the context of the protein folding energy landscape. Quantitatively, in principle, the diffusion-collision model for the building block association can be used. However, the folding rates of the building blocks and traps in their formation and association, need to be considered.     

Clustering of protein structural fragments reveals modular building block approach of nature

Structures of peptide fragments drawn from a protein can potentially occupy a vast conformational continuum. We co-ordinatize this conformational space with the help of geometric invariants and demonstrate that the peptide conformations of the currently available protein structures are heavily biased in favor of a finite number of conformational types or structural building blocks. This is achieved by representing a peptides' backbone structure with geometric invariants and then clustering peptides based on closeness of the geometric invariants. This results in 12,903 clusters, of which 2207 are made up of peptides drawn from functionally and/or structurally related proteins. These are termed "functional" clusters and provide clues about potential functional sites. The rest of the clusters, including the largest few, are made up of peptides drawn from unrelated proteins and are termed "structural" clusters. The largest clusters are of regular secondary structures such as helices and beta strands as well as of beta hairpins. Several categories of helices and strands are discovered based on geometric differences. In addition to the known classes of loops, we discover several new classes, which will be useful in protein structure modeling. Our algorithm does not require assignment of secondary structure and, therefore, overcomes the limitations in loop classification due to ambiguity in secondary structure assignment at loop boundaries.     

Comparison of intramolecular and intermolecular reactions in protein folding

The intrinsic component of the standard free energy change for the formation of a disulfide bond in a protein molecule is compared to that for an analogous chemical reaction. The former reaction, which represents theintramolecular formation of a disulfide bond in a protein molecule from a cysteine group containing a mixed disulfide bond with glutathione, and a free cysteine residue, is a unimolecular reaction. In contrast, its chemical analogue is a bimolecular reaction, and corresponds to theintermolecular disulfide interchange between a mixed disulfide-bonded compound between a cysteine residue and glutathione, and a free cysteine molecule. The difference in the intrinsic free energy of the above two reactions is estimated by two different approaches. First, a theoretical estimate of the magnitude of the difference in free energy of the two reactions (for a standard state of 1 M) is obtained using a gas-phase statistical thermodynamic approach, which indicates that the intramolecular reaction is energetically favored over its intermolecular counterpart by as much as 15.6 kcal/mole. For comparison, an experimentally derived value is also obtained, using experimental data from a study by Konishi et al. of the regeneration of the protein ribonuclease A (RNase A) from its reduced form by reduced and oxidized glutathiones. The intrinsic component of the free energy change of the intramolecular reaction, as it occurs in the protein molecule, is obtained from such experimental data by accounting explicitly for the free energy change (assumed to be solely an entropy change) pertaining to the conformational changes (ring closure) that the protein molecule undergoes in the course of the reaction. On the basis of the value derived from such an experimental approach, the intramolecular reaction is also energetically more favorable as compared to its intermolecular analogue, but only by a difference of 2.3 kcal/mole (for a standard state of 1 M). The large apparent discrepancy between the two values estimated from the theoretical and experimental approaches is rationalized by the postulation of several additional factors not inherent in the gas-phase theoretical estimate, such as dehydration and intramolecular hydrogen-bonding effects, which can largely compensate for the otherwise favorable energetics of the intramolecular reaction.     

Effective protein folding in simple random search

Here I show the following facts using a simple random search model without including any sophisticated energy term. As the size of elements exponentially affects the efficiency of folding, it can be remarkably enhanced by dividing the elements into small blocks. As the folding of the blocks is completely independent, the total folding time can be reduced to the folding time of the single hardest block. This result gives the simplest and most straightforward answer to the Levinthal paradox.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Computer building and folding of fictitious transfer-RNA sequences

In order to evaluate the common occurrence with which polynucleotides may adopt the cloverleaf configuration, 1150 random sequences were computer built and folded into their most stable secondary structure. Various constraints modulated the generation of the sequences: i) the base-pairing pattern, ii) the nucleotide composition, iii) the presence of assigned bases (modified or not) at certain sites, and iv) the chain length. In many cases, artificial tRNAs appear to require a more complex organization than a cloverleaf pairing scheme to achieve, as do natural molecules, the corresponding secondary structure. Moreover, the preferred foldings of sequences from 50 to 90 nucleotide long without an imposed pairing pattern usually contain two rather than three hairpin-loops. Implications concerning the emergence and the evolution of the protein-synthesis apparatus are discussed.     

Synthesis of a GlcNAcylated arginine building block for the solid phase synthesis of death domain glycopeptide fragments

Herein we describe the synthesis of glycopeptide fragments from the death domains of TRADD and FADD bearing the recently discovered Nω-GlcNAc-β-arginine post-translational modification. TRADD and FADD glycopeptides were accessed through the use of a suitably protected synthetic glycosylamino acid ‘cassette’ that could be directly incorporated into conventional solid phase peptide synthesis (SPPS) protocols.     

Effect of osmolytes and chaperone-like action of P-protein on folding of nucleocapsid protein of Chandipura virus.

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such as d-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells.     

Analysis of fragments induced by simulated lattice protein folding

The folding process of a set of 42 proteins, representative of the various folds, has been simulated by means of a Monte Carlo method on a discrete lattice, using two different potentials of mean force. Multiple compact fragments of contiguous residues are formed in the simulation, stable in composition, but not in geometry. During time, the number of fragments decreases until one final compact globular state is reached. We focused on the early steps of the folding in order to evidence the maximum number of fragments, provided they are sufficiently stable in sequence. A correlation has been established between these proto fragments and regular secondary-structure elements, whatever their nature, alpha helices or beta strands. Quantitatively, this is revealed by an overall mean one-residue quality factor of nearly 60%, which is better for proteins mainly composed of alpha helices. The correspondence between the number of fragments and the number of secondary-structure elements is of 77% and the regions separating successive fragments are mainly located in loops. Besides, hydrophobic clusters deduced from HCA correspond to fragments with an equivalent accuracy. These results suggest that folding pathways do not contain structurally static intermediate. However, since the beginning of folding, most residues that will later form one given secondary structure are kept close in space by being involved in the same fragment. This aggregation may be a way to accelerate the formation of the native state and enforces the key role played by hydrophobic residues in the formation of the fragments, thus in the folding process itself.     

Independent folding of autolytic fragments of thermolysin and their domain-like properties

High molecular weight autolysis products from thermolysin have been isolated and identified. The primary fragments correspond to residues 1 to 187-204 (21kD) and residues 187-204 to 316 (12kD), respectively. The fragments are both capable of independent refolding upon removal of denaturant. On the basis of these results, we suggest that the first step in the unfolding pathway of thermolysin involves unfolding of an interdomain region and domain separation. Bound calcium ions at sites 1, 2 and 4 play a major role in protecting the protein against both autolysis and unfolding, probably by stabilizing the interdomain region and enhancing domain-domain interactions.     

Structure and folding of potato type II proteinase inhibitors: circular permutation and intramolecular domain swapping

Potato type II serine proteinase inhibitors are proteins that consist of multiple sequence repeats, and exhibit a multidomain structure. The structural domains are circular permutations of the repeat sequence, as a result of intramolecular domain swapping. Structural studies give indications for the origins of this folding behaviour, and the evolution of the inhibitor family.     

Replication: DNA building block synthesis on demand

Correct regulation of DNA nucleotide biosynthesis is emerging as a key issue of importance for genome integrity. The fission yeast Spd1 protein can modulate the activity of ribonucleotide reductase (RNR) by at least three different mechanisms. Now a paper reports that Spd1 turnover is linked to ongoing DNA synthesis.     

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.     

Binding and folding of melittin in the presence of ganglioside GM1 micelles

In this work we examine the binding and folding of the membrane-active peptide, melittin in the presence of ganglioside GM1 micelle. The membrane bilayer is capable of inducing folding to small proteins and peptides upon binding. Using two-dimensional NMR techniques we have shown that at low concentration, GM1 micelle is able to induce an extended helical conformation to MLT. The pulsed-field gradient diffusion NMR study indicates that the peptide partition into GM1 micelle along with about 32% binding. While looking for the binding between MLT and GM1 using saturation transfer difference NMR spectroscopy, Val5, Leu9, Thr11, Ile17, Ser18, and Trp19 have been identified as the residues that are in close proximity to GM1 micelles.     

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.     

Microbial production of building block chemicals and polymers

Owing to our increasing concerns on the environment, climate change, and limited natural resources, there has recently been considerable effort exerted to produce chemicals and materials from renewable biomass. Polymers we use everyday can also be produced either by direct fermentation or by polymerization of monomers that are produced by fermentation. Recent advances in metabolic engineering combined with systems biology and synthetic biology are allowing us to more systematically develop superior strains and bioprocesses for the efficient production of polymers and monomers. Here, we review recent trends in microbial production of building block chemicals that can be subsequently used for the synthesis of polymers. Also, recent successful cases of direct one-step production of polymers are reviewed. General strategies for the production of natural and unnatural platform chemicals are described together with representative examples.     

Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding

Applications of sensitive new technologies, in particular, two-dimensional NMR spectroscopy, have allowed detection of folded structures in short peptide fragments of proteins in aqueous solution under conditions where native proteins fold. These structures are in rapid dynamic exchange with unfolded states. These observations provide evidence in support of models for protein folding which postulate localized regions of folded structure as initiation sites for the folding process. Since these initiation processes are expected to be rapid, such models are consistent with kinetic evidence that the rate-determining steps of protein folding occur late in the process and probably involve rearrangement of incorrectly folded intermediates.     

Determination of intramolecular distance distribution during protein folding on the millisecond timescale

A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed. The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined. The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument). At that time the width of that distribution was rather large (16+/-2 A). The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10(-7) cm(2)/second. In a second slower phase of the refolding transition, the width was reduced to its native value (6+/-4 A).     

Binding characteristics of complementary fibronectin fragments on artificial substrata

Various properties have been evaluated for the binding to tissue culture substrata of proteolytic fragments of human plasma or cellular fibronectins containing complementary sequences from the individual and alternatively spliced chains, since related fragments are known to yield differing adhesive responses from cells. These studies utilize ELISA methods and a polyclonal antiserum directed to human pFN for direct measurement or an occupancy test utilizing anti-albumin. Very related fragments (with or without an extra type III homology unit or extra domaina or b) have significantly different properties in substratum binding and such differences provide a partial explanation for alteration of cellular adhesive responses on such fragments.     

Protein folding: binding of conformationally fluctuating building blocks via population selection.

Here we review different aspects of the protein folding literature. We present a broad range of observations, showing them to be consistent with a general hierarchical protein folding model. In such a model, local relatively stable, conformationally fluctuating building blocks bind through population selection, to yield the native state. The model includes several components: (1) the fluctuating building blocks that constitute local minima along the polypeptide chain, which even if unstable still possess higher population times than all alternate conformations; (2) the landscape around the bottom of the funnels; (3) the consideration that protein folding involves intramolecular recognition; (4) similar landscapes are observed for folding and for binding, and that (5) the landscape is dynamic, changing with the conditions. The model considers protein folding to be guided by native interactions. The reviewed literature includes the effects of changing the conditions, intermediates and kinetic traps, mutations, similar topologies, fragment complementation experiments, fragments and pathways, focusing on one specific well-studied example, that of the dihydrofolate reductase, chaperones, and chaperonines, in vivo vs. in vitro folding, still using the dihydrofolate example, amyloid formation, and molecular "disorder". These are consistent with the view that binding and folding are similar events, with the differences stemming from different stabilities and hence population times.