600 research outputs found
Constructing ensembles for intrinsically disordered proteins
The relatively flat energy landscapes associated with intrinsically disordered proteins makes modeling these systems especially problematic. A comprehensive model for these proteins requires one to build an ensemble consisting of a finite collection of structures, and their corresponding relative stabilities, which adequately capture the range of accessible states of the protein. In this regard, methods that use computational techniques to interpret experimental data in terms of such ensembles are an essential part of the modeling process. In this review, we critically assess the advantages and limitations of current techniques and discuss new methods for the validation of these ensembles
STRUCTURAL INSIGHTS INTO FOLDED, UNFOLDED AND NASCENT PROTEIN STATES USING ENSEMBLE SAMPLING AND CLUSTER EXPANSION
Ph.DDOCTOR OF PHILOSOPH
Protein folding, metal ions and conformational states: the case of a di-cluster ferredoxin
Dissertation presented to obtain the PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaMetal ions are present in over thirty percent of known proteins. Apart from a well
established function in catalysis and electron transfer, metals and metal centres
are also important structural elements which may as well play a key role in
modulating protein folding and stability. In this respect, cofactors can act not only
as local structural stabilizing elements in the native state, contributing to the
maintenance of a given specific structural fold, but may also function as potential
nucleation points during the protein folding process...Fundação para a Ciência e Tecnologia is acknowledged for financial support, by
awarding a PhD Grant SFRH/BD/18653/2004. This work has been funded by the projects POCTI/QUI/37521; POCTI/QUI/45758
and PTDC/QUI/70101 all to Cláudio M. Gomes
Synthesis, conformational investigations and applications of alpha-peptides containing cis-beta-aminocyclopropane dicarboxylic acids
In this work different alpha-peptides containing two enantiomers of cis-β-amino cyclopropane carboxylic acid (β-ACC) were synthesized in order to extend our knowledge regarding their conformational preferences. Alternated peptides alpha-L-Xaa-β-ACC were synthesized and their conformations were investigated by IR, CD and NMR spectroscopy. Short alpha-L-Ala-β-ACC alternated peptides in a polar solvent like methanol adopt well defined helical secondary structures. When one enantiomer was incorporated, the peptides showed a 313 helix turn involving a hydrogen-bond between the NH of the first β-ACC and the C=O of the following β-ACC unit. The CD spectrum corresponding to this structure shows a minimum at about 200 nm. CD spectra, which were measured in different solvents such as trifluoroethanol or hexafluoroaceton and in methanol with different amounts of water, suggest that the stability of the 313 helix-turn is strongly dependent on the environment.
The NMR data obtained from the analogues containing the other enantiomer of β-ACC display a structure, which is less well defined. There is likely a set of conformers which rapidly interconvert on the NMR timescale. CD investigations in different solvents show patterns that are almost solvent independent. The solvents investigated are probably not able to stabilize one conformation with respect to another one.
In the second part of this PhD work, β-ACC units were used as building blocks in biological active analogues of neuropeptide Y and RGD peptides, inducing a particular spatial orientation of important side chains for the interaction. Some β-ACC containing peptides are found to be able to enhance the biological affinity to the corresponding receptor. Conformational investigation on them allowed us to collect more information about the active conformation being responsible for the binding or at least on the conformation that is recognized by the receptor.
Cyclic pentapeptides containing the typical integrin recognition sequence Arg-Gly-Asp were synthesized and their affinities to the alphaVbeta3 receptor were investigated. The alphaVbeta3 integrin receptor subtype plays an important role in many physiological events and it is a potential pharmaceutical target for treatment of several diseases including tumors.
The stereochemistry of the β-ACC moiety is decisive for the receptor affinity. One pentapeptide is found to have a higher affinity compared to the reference peptide c(Arg-Gly-Asp-D-Phe-Val).
Conformational NMR investigation on this peptide shows the Arg-Gly-Asp segment in the positions i to i+2 of a type II� β-turn in which the β-ACC moiety occupies the position i+3.
The introduction of the β-ACC moieties in the C-terminal dodecapeptide of the NPY, at the position 10 (position 34 corresponding to the full length NPY) led to a shorter NPY analogue which is able to bind the Y1 NPY receptor subtype with good affinity and selectivity.
The secondary structure of the most selective analogue was investigated by 2D NMR in the presence of micelles that mimic the cell membrane, in order to gain information about the conformation that is recognized by the Y1 receptor subtype after the absorption step on the cell membrane. The observed secondary structure, differs substantially from the conformation present in the C-terminal part of the full length NPY, both from the helical structure found in solution in the presence of micelles, and from the typical PP-fold that has a flexible C-terminus.
The N-terminus of the peptide shows an amphiphilic helix that exposes hydrophobic side chains on one side, presumably forming the micelle-binding interface. At the C-terminus, the β-ACC moiety interrupts the helix and induced a turn-like structure. This conformation could be responsible for a particular orientation of the key side chains for the receptor recognition leading consequently to receptor selectivity
Alterações funcionais e conformacionais promovidas numa proteinase aspártica por trifluoroetanol
Tese de doutoramento em Bioquímica (Enzimologia), apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraO estudo das proteinases aspárticas tem vindo a ganhar interesse
devido à importância desta classe de enzimas na etiologia
e evolução de doenças humanas que são hoje fonte duma preocupação
crescente, como são os casos da doença de Alzheimer,
do cancro da mama ou da SIDA.
A base molecular de algumas destas doenças está associada a
erros de folding (enrolamento), que impossibilitam a sua função.
Estudos de estabilidade sobre esta classe de enzimas são de
extrema importância para a caracterização dos mecanismos patológicos
envolvidos, e para a descoberta de soluções terapêuticas.
A proteína heterodimérica cardosina A é uma proteinase aspártica
de origem vegetal que pode ser purificada em elevadas
quantidades. Apesar de tradicionalmente ser usada como agente
coagulante do leite na produção de queijo, as suas características
fizeram dela uma enzima interessante para aplicações biotecnológicas. Esta endopeptidase tem sido considerada um bom modelo
para estudos estruturais e funcionais do grupo das proteinases
aspárticas e de proteínas em geral.
A disponibilidade de água é um factor fundamental para a
estabilidade proteica e para a sua flexibilidade conformacional,
características estas essenciais à sua função.
Com o presente trabalho esperamos ter contribuído para a
compreensão da conformação nativa das proteinases aspárticas
como uma forma mutável. Ao longo dos últimos anos a estrutura
conformacional da cardosina A foi caracterizada em sistemas
bifásicos, meio aquoso saturado com solventes orgânicos, e
mais exaustivamente na presença do solvente orgânico acetonitrilo.
Na sequência do trabalho desenvolvido pelo nosso grupo,
o unfolding (desenrolamento) da cardosina A foi induzido pelo
2,2,2-trifluoroethanol (TFE), um solvente orgânico polar e prótico
com propriedade muito diferentes dos solventes orgânicos previamente
usados. As características do TFE promovem alterações
distintas na estrutura da água, interagindo de um modo particular
com a camada de hidratação da proteína e com a própria
estrutura proteica. Além disso o TFE é conhecido por estabilizar
conformações complexas em vez de induzir a desnaturação.
Foi objectivo do presente trabalho seguir as alterações conformacionais
e funcionais promovidas pela proximidade do TFE à
estrutura da cardosina A. A dependência da função enzimática
em relação à estrutura proteica foi relacionada com a disponibilidade
de água e/ou de pontes de hidrogénio na superfície da
proteína. Diferentes métodos espectroscópicos (dicroísmo circular
e fluorescência intrínseca), medições de actividade enzimática, e
análise calorimétrica foram utilizados para detectar e caracterizar
os estados induzidos pelo solvente orgânico.
Finalmente foram aplicadas ao sistema simulações de dinâmica molecular/mecânica molecular (MD/MM) de forma a compreender
a interacção entre a proteína e as moléculas de solvente.
Os ensaios in vitro com a cardosina A em TFE promoveram
variações de folding dependentes da concentração do álcool.
Concentrações de TFE inferiores a 4% diminuíram a estabilidade
proteica, mas aumentaram reversivelmente a actividade enzimática.
Concentrações superiores a 20% de TFE no meio inactivaram
irreversivelmente a enzima e desenrolaram a sua estrutura
terciária, enquanto o seu conteúdo secundário em hélices foi
progressivamente aumentado (principalmente de segmentos
sem estruturação prévia). Por último, concentrações superiores
a 70% de TFE no meio inactivaram a enzima e promoveram um
vasto aumento na complexidade estrutural, na forma típica de
estruturas helicoidais abertas, alterações estas que provaram ser
reversíveis.
As simulações de MD com TFE e água, descreveram alterações
locais de flexibilidade proteica, mas sem grandes transformações
conformacionais. Em vez disso o modelo expôs os locais de
competição entre o TFE e as moléculas de água da superfície
de solvatação. Moléculas de TFE foram encontradas a substituir
várias moléculas de hidratação no local activo. Apesar da molécula
de água catalítica não ter sido perdida na última conformação
adquirida na simulação para alto conteúdo em TFE, o local activo
apresentava-se ocupado por várias moléculas de TFE, e este
facto foi proposto como justificação para a perda de actividade. A
mesma lógica poderá explicar a recuperação de actividade após
a diluição para sistema aquoso, com a libertação do local activo
para interacção com o substrato.The study of aspartic proteases has been gaining interest due
to their importance in the development of major concerning
human diseases, as Alzheimer’s disease, breast cancer, or AIDS.
The molecular basis of some of these diseases is associated
to folding errors, which disables proteins proper functioning.
Stability studies over this class of enzymes are extremely important
for characterising the pathology involved mechanisms and
to discover therapeutic solutions.
The heterodimeric cardosin A is a plant aspartic proteinase of
high yield purification. Besides having been traditionally used as
milk clotting agent for cheese making, its characteristics have
made it an interesting enzyme for biotechnological applications.
This endopeptidase has thus been considered a good model
for structural and functional studies of the aspartic proteinases
group, and of proteins in general. Water availability is a fundamental factor for protein stability and
conformation flexibility, and these characteristics are imperative
for proper functioning. The present approach intends to upgrade
the understanding of aspartic proteases native conformation as
a mutable form. Along the last few years, cardosin A structural
conformation has been characterized in biphasic systems, aqueous
solutions saturated by organic solvents, and more extensively in
the presence of the organic solvent acetonitrile. In sequence with
the work developed by our group, the unfolding of cardosin A was
here induced by 2,2,2-trifluoroethanol (TFE), a polar and protic
organic solvent with very different properties from the previous
organic solvents tested. TFE characteristics promote distinct
alterations in water structure, interacting in a particular way with
the protein hydration layer and with the protein structure itself.
Furthermore, it is known to stabilize well ordered conformations
rather than inducing denaturation.
The aim of the present work was to follow the conformational
and functional alterations promoted by TFE proximity to cardosin
A structure. The function dependence on enzyme structure was
related to the availability of water and/or of hydrogen bonds to
the protein surface. Different spectroscopic methods (circular
dichroism and intrinsic fluorescence), activity measurements,
and calorimetric analysis were employed to detect and characterize
the organic solvent induced states. Finally, molecular
dynamics/molecular mechanics (MD/MM) simulations were
applied to the system in order to understand the interaction
between protein and solvent molecules.
The TFE in vitro assays with cardosin A promoted folding variations
dependent of the alcohol concentration. TFE medium
content below 4% decreased protein stability, but reversibly
increased its enzymatic rate. TFE medium content over 20%. irreversibly inactivated the enzyme and unfolded its tertiary
structure, while secondary helical content was progressively
increased (mainly from previously unordered segments). At
last, TFE medium content over 70% inactivated the enzyme
and promoted a vast increase in structural complexity, taking
form as characteristic open helical structures, and these alterations
proved to be reversible.
MD simulations with TFE and water described local alterations in
protein flexibility, but no large conformational transformations.
Instead, the model described an exposition of local competition
of TFE with water for solvation surface. TFE molecules were
found replacing several hydration molecules in the active site.
Despite the catalytic water was not lost in the last acquired
conformation of the high TFE content MD simulation, the active
site was occupied by several TFE molecules, and this occurrence
was proposed to justify the activity loss. The same reasoning
can explain the activity recovery upon aqueous dilution, with
the release of the active site for substrate binding.Fundação para a Ciência e a Tecnologia,
Portugal (projecto POCTI/QUI/60791/2004 e bolsa de doutoramento SFRH/BD/10754/2002
The Eighth Central European Conference "Chemistry towards Biology": snapshot
The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August – 1 September 2016The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August-1 September 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting
Unfolding mechanism of human glutathione transferase M1a-1a
A thesis submitted to the Faculty of Science, University of the Witwatersrand,
Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy
May 2018Proteins exist in equilibrium between the native (N) and the denatured (D) states. In order to form the biologically active native state, the amino acid sequence has to fold to form a stable three-dimensional structure. The large scientific community of biochemists and biophysicists has not yet been able to gain a complete understanding of this process. In this study, the unfolding of the homodimeric detoxification enzyme hGST M1a-1a (WT dimer) was investigated. Additionally, an F56S/R81A double-mutant (mutant monomer) was engineered to create a monomeric form of the protein. The mutant monomer was used to gain a better understanding of the unfolding events occurring at the subunit level, in the absence of quaternary interactions. Data from various techniques indicate the mutant monomer to closely resemble the tertiary structure of the subunits in the WT homodimer, making it a suitable model to study the unfolding mechanism of hGST M1a in the absence of quaternary interactions. A four-state equilibrium unfolding mechanism, involving two stable intermediate species, is proposed. HDX-MS studies indicate that disruption of the conserved lock-and-key motif, as well as the structures surrounding the mu loop, results in a destabilisation of domain 1. However, dimer dissociation cannot occur until the mixed charge cluster at the dimer interface has been destabilised. The destabilisation of domain 1 results in destabilisation of α4 and α5 in domain 2, because the domains unfold in a concerted manner. hGST M1a-1a dissociates to form monomeric intermediate (M), with weak interdomain interactions and compromised short-range contacts. The unstable M intermediate self-associates to form an oligomeric intermediate (I). The destabilisation of α6 and α7 in the hydrophobic core of domain 2 drives the formation of the partially structured denatured state. Further investigation will need to be pursued to determine whether hGST M1a-1a unfolds via transient intermediate states; however, the elucidation of the equilibrium unfolding pathway of a complex homodimeric protein is a valuable addition to the ever-growing knowledge base of protein folding.MT 201
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