The denatured state of N-PGK is compact and predominantly disordered

The Organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I-b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I-k) and denatured (D)]. The transition to I-b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I-b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I-b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics Of I-b indicate that it forms from the fully denatured state within 100 mu s, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I-b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology. (C) 2008 Elsevier Ltd. All rights reserved

The difficulty of folding self-folding origami

Why is it difficult to refold a previously folded sheet of paper? We show that even crease patterns with only one designed folding motion inevitably contain an exponential number of `distractor' folding branches accessible from a bifurcation at the flat state. Consequently, refolding a sheet requires finding the ground state in a glassy energy landscape with an exponential number of other attractors of higher energy, much like in models of protein folding (Levinthal's paradox) and other NP-hard satisfiability (SAT) problems. As in these problems, we find that refolding a sheet requires actuation at multiple carefully chosen creases. We show that seeding successful folding in this way can be understood in terms of sub-patterns that fold when cut out (`folding islands'). Besides providing guidelines for the placement of active hinges in origami applications, our results point to fundamental limits on the programmability of energy landscapes in sheets.Comment: 8 pages, 5 figure

Directionality in protein fold prediction

<p>Abstract</p> <p>Background</p> <p>Ever since the ground-breaking work of Anfinsen et al. in which a denatured protein was found to refold to its native state, it has been frequently stated by the protein fold prediction community that all the information required for protein folding lies in the amino acid sequence. Recent in vitro experiments and in silico computational studies, however, have shown that cotranslation may affect the folding pathway of some proteins, especially those of ancient folds. In this paper aspects of cotranslational folding have been incorporated into a protein structure prediction algorithm by adapting the Rosetta program to fold proteins as the nascent chain elongates. This makes it possible to conduct a pairwise comparison of folding accuracy, by comparing folds created sequentially from each end of the protein.</p> <p>Results</p> <p>A single main result emerged: in 94% of proteins analyzed, following the sense of translation, from N-terminus to C-terminus, produced better predictions than following the reverse sense of translation, from the C-terminus to N-terminus. Two secondary results emerged. First, this superiority of N-terminus to C-terminus folding was more marked for proteins showing stronger evidence of cotranslation and second, an algorithm following the sense of translation produced predictions comparable to, and occasionally better than, Rosetta.</p> <p>Conclusions</p> <p>There is a directionality effect in protein fold prediction. At present, prediction methods appear to be too noisy to take advantage of this effect; as techniques refine, it may be possible to draw benefit from a sequential approach to protein fold prediction.</p

Biologic Constraints on Modelling Virus Assembly

The mathematic modelling of icosahedral virus assembly has drawn increasing interest because of the symmetric geometry of the outer shell structures. Many models involve equilibrium expressions of subunit binding, with reversible subunit additions forming various intermediate structures. The underlying assumption is that a final lowest energy state drives the equilibrium toward assembly. In their simplest forms, these models have explained why high subunit protein concentrations and strong subunit association constants can result in kinetic traps forming off pathway partial and aberrant structures. However, the cell biology of virus assembly is exceedingly complex. The biochemistry and biology of polyoma and papillomavirus assembly described here illustrates many of these specific issues. Variables include the use of cellular ‘chaperone’ proteins as mediators of assembly fidelity, the coupling of assembly to encapsidation of a specific nucleic acid genome, the use of cellular structures as ‘workbenches’ upon which assembly occurs, and the underlying problem of making a capsid structure that is metastable and capable of rapid disassembly upon infection. Although formidable to model, incorporating these considerations could advance the relevance of mathematical models of virus assembly to the real world

Insight into the multicopper oxidases stability

Dissertation presented to obtain the PhD degree in BiochemistryThis dissertation portrays recent development on the knowledge of the stability determinants and of functional characteristics of multicopper oxidases (MCO). Multicopper oxidases are a family of enzymes that includes laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2), ascorbate oxidase (L-ascorbate oxygen oxidoreductase, EC 1.10.3.3) and ceruloplasmin (Fe2+ oxygen oxidoreductase, EC 1.16.3.1). MCO are characterized by having four copper ions that are classified into three distinct types of copper sites, namely type 1 (T1), type 2 (T2) and type 3 (T3). The classical T1 copper site comprises two histidine residues and a cysteine residue arranged in a distorted trigonal geometry around the copper ion with bonding distances approx. 2.0 Å (1 Å=0.1 nm); a weaker fourth methionine ligand completes the tetrahedral geometry. The copper–cysteine linkage is characterized by an intense S(π)→Cu(dx2−y2) CT (charge transfer) absorption band at approximately 600 nm, and a narrow parallel hyperfine splitting A\\ = (43–90)×10−4 cm−1 in the electron paramagnetic resonance (EPR) spectrum. The function of the T1 copper site is to shuttle electrons from substrates to the trinuclear copper centre where molecular oxygen is reduced to two molecules of water during the complete four-electron catalytic cycle. The trinuclear center contains a T2 copper coordinated by two histidine residues and one water molecule, lacks strong absorption bands and exhibits a large parallel hyperfine splitting in the EPR spectrum (A\\ = (150–201)×10−4 cm−1). The T2 copper site is in close proximity to two T3 copper ions, which are each coordinated by three histidine residues and typically coupled, for example, through a dioxygen molecule. The T3 or coupled binuclear copper site is characterized by an intense absorption band at 330 nm originating from the bridging ligand and by the absence of an EPR signal due to the antiferromagnetically coupling of the copper ions.(...)Apoio financeiro da FCT e do FSE no âmbito do Quadro Comunitário de Apoio, BD nº SFRH/BD/31444/200

Reactions between Zinc Metallothionein and Carbonic Anhydrase

More than 25% of proteins require metal ion cofactors for structure or function. The interactions between metalloproteins have largely been overlooked, though these interactions ultimately govern metal localization and control metal ion homeostasis. Mammalian metallothionein (MT) is a small, cysteine-rich metalloprotein that binds numerous metal ions per protein strand. Up to seven divalent metals, such as zinc or cadmium, are wrapped into a clustered two-domain structure. This unusually high metal content places MT as an attractive candidate for studying interactions with other metal-binding proteins. This present study investigates the metal transfer reactions between MTs and other metalloproteins, using carbonic anhydrase (CA) as a putative zinc-dependent enzyme. This thesis presents electrospray ionization mass spectrometric (ESI-MS) data showing the competitive zinc metallation reactions between apoCA and various apoMTs. Modelling of the ESI-MS data is used to determine the reaction parameters and those parameters are shown to be reflected directly in the raw data. These results demonstrate how MT can act as a homeostatic buffer of metal ions, by binding them with different affinities. The kinetics of the metal transfers between zinc MTs and cadmium or zinc CA show that the rates of metal transfer between the two metalloproteins is directly dependent on the metal content of the MT. Further studies on the domain specific properties of MT using shortened MT domain fragment proteins show that: (i) there is no significant degree of domain specificity in metal binding to apoMTs; (ii) the weakest bound metal ion is located within the N-terminal domain of the intact MT protein; (iii) the highest affinity binding site is located within the C-terminal domain; and, (iv) domain-domain interactions within the MT peptide strand modulate metal binding affinities. Taken together, these results support the homeostatic roles of metallothionein proteins while also challenging the mechanisms for metal binding and release to apoenzymes

The Levinthal paradox: yesterday and today

A change in the perception of the protein folding problem has taken place recently. The nature of the change is outlined and the reasons for it are presented. An essential element is the recognition that a bias toward the native state over much of the effective energy surface may govern the folding process. This has replaced the random search paradigm of Levinthal and suggests that there are many ways of reaching the native state in a reasonable time so that a specific pathway does not have to be postulated. The change in perception is due primarily to the application of statistical mechanical models and lattice simulations to protein folding. Examples of lattice model results on protein folding are presented. It is pointed out that the new optimism about the protein folding problem must be complemented by more detailed studies to determine the structural and energetic factors that introduce the biases which make possible the folding of real proteins