Search

The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD) simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an "activated" form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins.

Flavodoxins are bacterial electron transport proteins whose redox competence is due to the presence of a tightly but noncovalently bound FMN molecule. While the thermodynamics of the complex are understood, the mechanism of association between the apoflavodoxin and the redox cofactor is not so clear. We investigate here the mechanism of FMN binding to the apoflavodoxin from Helicobacter pylori, an essential protein that is being used as a target to develop antimicrobials. This flavodoxin is structurally peculiar as it lacks the typical bulky residue interacting with the FMN re face but bears instead a small alanine. FMN binding is biphasic, regardless of the presence of phosphate molecules in solution, while riboflavin binding takes place in a single step, the rate constant of which coincides with the fast phase of FMN binding. A mutational study at the isoalloxazine and phosphate subsites for FMN binding clearly indicates that FMN association is always limited by interaction with the isoalloxazine subsite because mutating residues that interact with the phosphate moiety of FMN in the native complex hardly changes the observed rate constants and amplitudes. In contrast, replacing tyr92, which interacts with the isoalloxazine, greatly lowers the rate constants. Our analysis indicates that the two FMN binding phases observed are related neither with alternative or sequential interaction with the two binding subsites nor with the presence of bound phosphate. It is possible that they reflect the intrinsic conformational heterogeneity of the apoflavodoxin ensemble.

Intermediate conformations are crucial to our understanding of how proteins fold into their native structures and become functional. Conventional spectroscopic measurements of thermal denaturation transitions allow the detection of equilibrium intermediates but often provide little structural detail; thus, application of more informative techniques is required. Here we used small-angle X-ray scattering (SAXS) to study the thermal denaturation of four variants of Anabaena PCC 7119 flavodoxin, including the wild-type apo and holo forms, and two mutants, E20K/E72K and F98N. Denaturation was monitored from changes in SAXS descriptors. Although the starting and final points of the denaturation were similar for the flavodoxin variants tested, substantial differences in the unfolding pathway were apparent between them. In agreement with calorimetric data, analysis of the SAXS data sets indicated a three-state unfolding equilibrium for wild-type apoflavodoxin, a two-state equilibrium for the F98N mutant, and increased thermostability of the E20K/E72K mutant and holoflavodoxin. Although the apoflavodoxin intermediate consistently appeared mixed with significant amounts of either native or unfolded conformations, its SAXS profile was derived from the deconvolution of the temperature-dependent SAXS data set. The apoflavodoxin thermal intermediate was structurally close to the native state but less compact, thereby indicating incipient unfolding. The residues that foster denaturation were explored by an ensemble of equilibrium ϕ-value restrained molecular dynamics. These simulations pointed to residues located in the cofactor and partner-protein recognition regions as the initial sites of denaturation and suggest a conformational adaptation as the mechanism of action in apoflavodoxin., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Increasing protein stability is interesting for practical reasons and because it tests our understanding of protein energetics. We explore here the feasibility of stabilizing proteins by replacing underexposed polar residues by apolar ones of similar size and shape. We have compared the stability of wild-type apoflavodoxin with that of a few carefully selected mutants carrying Y → F, Q → L, T → V or K → M replacements. Although a clear inverse correlation between native solvent exposures of replaced polar residues and stability of mutants is observed, most mutations fail to stabilize the protein. The promising exceptions are the two Q → L mutations tested, which characteristically combine the greatest reduction in polar burial with the greatest increase in apolar burial relative to wild type. Analysis of published stability data corresponding to a variety of mutant proteins confirms that, unlike Y → F or T → V replacements, Q → L mutations tend to be stabilizing, and it suggests that N → L mutations might be stabilizing as well. On the other hand, we show that the stability changes associated to the apoflavodoxin mutations can be rationalized in terms of differential polar and apolar burials upon folding plus a generic destabilizing penalty term. Simple equations combining these contributions predict stability changes in a large data set of 113 mutants (Y → F, Q → L or T → V) similarly well as more complex algorithms available on the Internet.

FtsH is a peculiar prokaryotic protease with low unfoldase activity. Different reports have proposed that FtsH substrates could be either tagged proteins or proteins of low stability. We show here that FtsH degradation of 31 point mutants of Anabaena apoflavodoxin is inversely proportional to their conformational stabilities, and that the same applies to other substrate proteins. In contrast, highly stable proteins such as GST and holoflavodoxin are not degraded at all. Attempts to identify sequence tags signaling for degradation in apoflavodoxin fragments have been unsuccessful. Apoflavodoxin adopts three conformations: native, partly unfolded and fully unfolded. It is revealing that degradation of the 31 variants is proportional to the molar fraction of fully unfolded molecules and inversely proportional to the fraction of stable apoflavodoxin molecules. This indicates that FtsH, rather than unfolding the protein, acts on the fraction that is already unfolded., (Copyright 2010 Elsevier Inc. All rights reserved.)

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Apoflavodoxin from Anabaena PCC 7119 is a 169 residue globular protein of known structure and energetics. Here, we present a comprehensive Phi-value analysis to characterize the structure of its transition state. A total of 34 non-disruptive mutations are made throughout the structure and a range of Phi-values from zero to one are observed. In addition, a small set of eight aliphatic small-to-large mutations have been introduced in the hydrophobic core of the protein and they have been analyzed to investigate the feasibility of stabilizing the unfolding transition state by creating new non-native interactions. We find that the transition state of apoflavodoxin (so far the largest protein subjected to Phi-analysis) is diffuse and that it can be stabilized by unspecific hydrophobic interactions that can speed up the folding reaction. The data gathered on the apoflavodoxin transition state are compared with results from experimental studies in other proteins to revisit the relationship between the native state topology and transition state structure.