Research Summary

The study of biologically relevant macromolecules has been for quite some time a challenging endeavor for researchers, who must exploit a variety of methodologies. In recent years, molecular modeling and simulation is the field of research that probably has more enthusiastically contributed with information at the atomic level.

In our group, we are very interested in the study of the dynamic properties of proteins and lipid bilayers under different conditions of temperature, pH, ionic strength, redox potential and solvent composition.

Inclusion of pH effects in molecular modelling of lipid bilayers

KTP-Membrane     The lipid bilayer is the basic structural component of biological membranes. Membrane protein function is often strongly affected by the chemical and physical properties of the bilayer, which in turn depend on the composition of lipids and their complex interactions at the molecular level. A detailed description of a lipid bilayer at the atomic level has to take in consideration all important factors that affect in some way the membrane behavior and stability. pH is recognizably one of these factors even though it is usually forgotten due to the high complexity in terms of modeling. Changes in pH are usually associated with (de)protonation in key titrable groups present in the polar head of some phospholipids that constitute the bilayer. The resulting changes in the electrostatic environment will influence strongly the very structure of the bilayer allowing for the appearance of certain phenomena, like lipid phase transition and microdomain formation. Our objective is to apply the constant-pH MD methodology to the study of lipid protonation in bilayers.

Cardiolipin-rich lipid bilayers: the "acid-anion" formation at neutral pH

Cardiolipin     Cardiolipins (CLs) constitute an exceptional class of lipids. Unlike most of the other lipid types, CLs are anionic lipids with four acyl chains. CLs have two acidic sites that can be ionized. The protonation state of CLs is still a matter of debate and both single and double charged CLs have been proposed. This uncertainty seems to arise from difficulties in determining its charge, since it depends on pH and on environmental factors such as the concentration of CLs. A high pKa2 (>7.0) has been proposed on a potentiometric titration and has been associated with an internal "acid-anion" conformation. This high pKa value, inevitably influences our understanding of its conformation in bilayers. These features are very important in the ability of CL to play its role in ATP synthesis. Disruption of the molecule's key features that allow for this mechanism can result in the inability for CL to perform its biological function. Inefficient CLs have been associated with the Barth's syndrome disease. Even though many of these theories and mechanisms have been proposed over 25 years ago, there are still many questions regarding the way lipids exchange protons in their bilayer environment. One major hurdle is that many of the studies performed are technically very difficult to perform in a biological membrane or, sometimes, even in a simple bilayer model. Molecular modeling has also not been able to help us rationalize these mechanisms mainly due to limitations in the available methodologies. Our approach aims to solve this problem.

Study of the folding properties of peptide dendrimers

Dendrimer     Peptide dendrimers are a specific kind of dendrimers which are formed by alternating functional amino acids with branching diamino acids (see Figure). One major advantage of peptide dendrimer over de novo linear peptides in mimicking protein structure/function is the ability to predict and control their folded structure. Most dendritic peptides are topologically constrained to adopt a more globular shape. In this way, simple protein-like structures can be created where functions such as catalysis or molecular recognition occur by constructive interactions between amino acids as in natural proteins.

Study of the pH induced misfolding and possible reversibility of the human prion protein

hPrP     The human prion protein (hPrP) is associated with the Creutzfeldt-Jakob disease and other amyloid diseases known as transmissible spongiform encephalopathies. The normal form of PrP is monomeric and solube but can be transformed into a misfolded β-rich form (see Figure) which aggregates and forms amyloid fibrils. This transition can be induced by decreasing the pH and is thought to be caused "in vivo" by the low pH of endosomes. We are currently studying the possible reversibility of this transition using constant-pH MD simulations of misfolded hPrP conformations at neutral pH.

Charge parameterization in united atom force fields using QM and MM methodologies

Cytc3     DesulfoVibrio Vulgaris Hildenborough cytochrome c3 is a small (aprox. 14 kDa) globular and monomeric tetraheme protein (see Figure). It is constituted by 107 residues plus four hemes covalently bound to cysteines in the polypeptide chain together with bis-histidinyl axial ligation. DvHc3 redox titration using constant-(pH/E) MD revealed very strong electrostatic interactions between the 4 heme groups in the protein, which should be very sensitive to charge parameterization. The objective of this project is to improve this charge parameterization using QM and QM/MM methodologies in order to successfully model the different redox states in constant-(pH,E) MD simulations of multihemic cytochromes

Selected Recent Research Highlights

Feb. 10, 2016

Our first letter came out in J. Chem. Theory Comput.  and is called: "pKa values of titrable amino acids at the water/membrane interface". IMHO, it is a clear breakthrough in the state-of-the-art of pKa estimations in lipidic environments.

Nov. 12, 2015

A new paper with the new CpHMD-L methodology came out in J. Chem. Theory Comput.  The paper is called: "Constant-pH MD Simulations of DMPA/DMPC Lipid Bilayers"

Aug. 04, 2015

Our FCT project on the CpHMD-L simulations of the pHLIP peptide was approved and we got funded for the next 3-4 years :) (PTDC/QEQ-COM/5904/2014)

Jan. 15, 2015

A collaboration with the Structure and Reactivity group here at CQB resulted in a very interesting publication in Mol. Pharamaceutics on the "Molecular details of INH-C10 binding to wt KatG and to its S315T mutant"

Oct. 30, 2014

A very important paper came out in J. Chem. Theory Comput.  on the "Treatment of ionic strength in biomolecular simulations of charged lipid bilayers"

Approved Projects

(Team Leader)


CpHMD-L simulations of pHLIP peptides: design of new tumor-targeted drug delivery systems


Adding realism to the molecular modeling of lipidic membranes: inclusion of pH effects

(Team Member)

CMST COST Action CM1102

Multivalent Glycosystems for Nanoscience - MultiGlycoNano (Management Committee)


Aumentando o realismo da modelação de membranas em métodos de dinâmica molecular a pH constante: inclusão de gradientes electroquímicos e titulação de lípidos

Bilateral Program: PESSOA

Complexos de metais de transição usados como intercaladores de DNA: investigação teórica. (Lisboa - Strasbourg)

Bilateral Program: FCT-CSIC

Síntese, estudos computacionais e propriedades biológicas de compostos de ouro (Lisboa - Zaragoza)


Understanding structure-activity relationships in peptide dendrimers using a molecular modelling approach


Including protonation effects in the simulation of peptides and proteins in membrane environments


Study of pH-dependent protein misfolding using state-of-the-art molecular modeling methods


  1. Jesus, A. R., Vila-Viçosa, D., Machuqueiro, M., Marques, A. P. S., Dore, T. M., and Rauter, A. P., (2017) "Targeting Type 2 Diabetes with C-Glucosyl Dihydrochalcones as Selective Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors: Synthesis and Biological Evaluation", J. Med. Chem., accepted. doi

  2. Filipe, L. C. S., Machuqueiro, M., Darbre, T., Baptista, A. M., (2016) "Exploring the Structural Properties of Positively Charged Peptide Dendrimers", J. Phys. Chem. B, 120, 11323-11330. doi

  3. Teixeira, V. H., Capacho, A. S. C., Machuqueiro, M., (2016) "The role of electrostatics in TrxR electron transfer mechanism: A computational approach", Proteins Struct. Funct. Bioinf., 84, 1836-1843. doi

  4. Filipe, L. C. S., Campos, S. R. R., Machuqueiro, M., Darbre, T., Baptista, A. M., (2016) "Structuring Peptide Dendrimers Through pH Modulation and Substrate Binding", J. Phys. Chem. B, 120, 10138-10152. doi

  5. Ressaissi, A., Attia, N., Falé, P. L. V., Pacheco, R., Teixeira, V. H., Machuqueiro, M., Borges, C., Serralheiro, M. L. M., (2016) "Aqueous Extracts from Nopal (Opuntia Ficus-Indica): Antiac-etylcholinesterase and Antioxidant Activity from Phenolic Bioactive Compounds", Int. J. Green Herb. Chem., 5, 337-348. link

  6. Teixeira, V. H., Vila-Viçosa, D., Reis, P. B. P. S., Machuqueiro, M., (2016) "pKa values of titrable amino acids at the water/membrane interface", J. Chem. Theory Comput., 12, 930-934. doi

  7. Vilas-Boas, F., Bagulho, A., Tenente, R., Teixeira, V. H., Martins, G., Costa, G., Jerónimo, A., Cordeiro, C., Machuqueiro, M., Real, C. (2016) "Hydrogen Peroxide regulates Cell Adhesion through the Redox Sensor RPSA", Free Radic. Biol. Med., 90, 145-157. doi

  8. Santos, H. A. F., Vila-Viçosa, D., Teixeira, V. H., Baptista, A. M., Machuqueiro, M. (2015) "Constant-pH MD simulations of DMPA/DMPC lipid bilayers", J. Chem. Theory Comput., 11, 5973-5979. doi

  9. Magalhães, P. R., Machuqueiro, M., Baptista, A. M. (2015) "Constant-pH molecular dynamics study of kyotorphin in an explicit bilayer", Biophys. J., 108, 2282-2290. doi

  10. Vila-Viçosa, D., Teixeira, V. H., Baptista, A. M., Machuqueiro, M., (2015) "Constant-pH MD simulations of an oleic acid bilayer", J. Chem. Theory Comput., 11, 2367-2376. doi

  11. Teixeira, V.H., Ventura, C., Leitão, R., Rafols, C., Bosch, E., Martins, F., Machuqueiro, M., (2015) "Molecular details of INH-C10 binding to wt KatG and to its S315T mutant", Mol. Pharmaceutics, 12, 898-909. doi

  12. Vila-Viçosa, D., Teixeira, V. H., Santos, H. A. F., Baptista, A. M., Machuqueiro, M., (2014) "Treatment of ionic strength in biomolecular simulations of charged lipid bilayers", J. Chem. Theory Comput., 10, 5483-5492. doi

  13. Mestre, A.S., Machuqueiro, M., Silva, M., Freire, R., Fonseca, I.M., Santos, S.C.S., Calhorda, M.J., Carvalho, A.P., (2014) "Influence of activated carbons porous structure on iopamidol adsorption", Carbon, 77, 607-615. doi

  14. Vila-Viçosa, D., Francesconi, O., Machuqueiro, M., (2014) "Why a diaminopyrrolic tripodal receptor binds mannosides in acetonitrile but not in water?", Beilstein J. Org. Chem., 10, 1513-1523. doi

  15. Teixeira, V. H., Vila-Viçosa, D., Baptista, A. M., Machuqueiro, M., (2014) "Protonation of DMPC in a bilayer environment using a linear response approximation", J. Chem. Theory Comput., 10, 2176-2184. doi

  16. Estácio, S. G., Krobath, H., Vila-Viçosa, D., Machuqueiro, M., Shakhnovich, E.I., Faísca, P.F.N., (2014), "A simulated intermediate state for folding and aggregation provides insights into ΔN6 β2-microglobulin amyloidogenic behavior", PLoS Comput. Biol., 10, e1003606. doi

  17. Filipe, L., Machuqueiro, M., Darbre, T., Baptista, A. M., (2013) "Unraveling the Conformational Determinants of Peptide Dendrimers Using Molecular Dynamics Simulations", Macromolecules, 46, 9427-9436. doi

  18. Carvalheda, C. A., Campos, S. R., Machuqueiro, M., Baptista, A. M., (2013) "Structural Effects of pH and Deacylation on Surfactant Protein C in an Organic Solvent Mixture: A Constant-pH MD Study", J. Chem. Inf. Model., 53, 2979-2989. doi

  19. Vila-Viçosa, D., Teixeira, V. H., Santos, H. A. F., Machuqueiro, M., (2013) "Conformational Study of GSH and GSSG Using Constant-pH Molecular Dynamics Simulations", J. Phys Chem. B, 117, 7507-7517. doi

  20. Martins, A., Santos, M. S., Dias, C., Serra, P., Cachatra, V., Pais, J., Caio, J., Teixeira, V. H., Machuqueiro, M., Silva, M. S., Pelerito, A., Justino, J., Goulart, M., Silva, F. V., Rauter, A. P. (2013) "Tuning bioactivity of new tensioactive deoxy glycosides with structure: antibacterial activity vs. selective cholinesterase inhibition rationalized by molecular docking", Eur. J. Org. Chem., 2013, 1448-1459. doi

  21. Henriques, J., Costa, P. J., Calhorda, M. J., Machuqueiro, M. (2013) "Charge Parametrization of the DvH-c3 Heme Group: Validation Using Constant-(pH,E) Molecular Dynamics Simulations", J. Phys. Chem. B, 117, 70-82. doi

  22. Vila-Viçosa, D., Campos, S.R.R., Baptista, A.M., Machuqueiro, M., (2012) "Reversibility of Prion Misfolding: Insights from Constant-pH Molecular Dynamics Simulations", J. Phys. Chem. B, 116, 8812-8821. doi

  23. Machuqueiro, M., Baptista, A.M. (2011) "Is the prediction of pKa values by constant-pH molecular dynamics being hindered by inherited problems?", Proteins: Struct. Funct. Bioinf., 79, 3437.doi

  24. Gamelas, C.A., Bandeira, N.A.G., Pereira, C.C.L., Calhorda, M.J., Herdtweck, E., Machuqueiro, M., Romão, C.C., Veiros, L.F. (2011) "Indenyl ring slippage in crown thioether complexes [IndMo(CO)2L]+ and C-S activation of trithiacyclononane: Experimental and theoretical studies", Dalton Trans., 40, 10513.doi

  25. Filipe, L.C.S., Machuqueiro, M., Baptista, A.M. (2011) "Unfolding the conformational behavior of peptide dendrimers: insights from molecular dynamics simulations", J. Am. Chem. Soc., 133, 5042-5052. doi

  26. Campos, S., Machuqueiro, M., Baptista, A.M. (2010) "Constant-pH molecular dynamics simulations reveal a β-rich form of the human prion protein", J. Phys. Chem. B, 114, 12692. doi

  27. Machuqueiro, M., Campos, S., Soares, C.M., Baptista, A.M. (2010) "Membrane-induced conformational changes of kyotorphin revealed by molecular dynamics simulations", J. Phys. Chem. B, 114, 11659-11667. doi

  28. Machuqueiro, M., Baptista, A.M. (2009) "Molecular dynamics at constant pH and reduction potential: application to cytochrome c3", J. Am. Chem. Soc., 131, 12586-12594. doi

  29. Machuqueiro, M., Baptista, A.M. (2008) "Acidic range titration of HEWL using a constant-pH molecular dynamics method", Proteins, 72, 289. doi

  30. Machuqueiro, M., Baptista, A.M. (2007) "The pH-dependent conformational states of kyotorphin: a constant-pH molecular dynamics study", Biophys. J., 92, 1836-45. doi

  31. Machuqueiro, M., Baptista, A.M. (2006) "Constant-pH Molecular Dynamics with ionic strength effects: The protonation-conformation coupling in decalysine", J. Phys. Chem. B, 110, 2927-2933. doi

  32. Teixeira, V.H., Cunha, C.A., Machuqueiro, M., Oliveira, A.S.F., Victor, B.L., Soares, C.M., Baptista, A.M. (2005) "On the use of different dielectric constants for computing individual and pairwise terms in Poisson-Boltzmann studies of protein ionization equilibrium", J. Phys. Chem. B, 109, 14691-706. doi

  33. Fernandez-Lopez, R., Kofoed, J., Machuqueiro, M., Darbre, T. (2005) "A selective direct aldol reaction in aqueous media catalyzed by zinc-proline", Eur. J. Org. Chem., 5268-76. doi

  34. Kofoed J, Machuqueiro, M., Reymond, J.-Louis, Darbre, T. (2004) "Zinc-proline catalyzed pathway for the formation of sugars", Chem. Comm., 1540-1. doi

  35. Darbre, T., Machuqueiro, M. (2003) "Zn-Proline catalyzed direct Aldol reaction in aqueous medium", Chem. Commun., 1090-1091. doi

  36. Machuqueiro, M., Darbre, T. (2003) "Zinc mediated methyl transfer from trimethyl phosphate to chelating and non-chelating thiols. Model for Zn-dependent methyltransferases", J. Inorg. Biochem., 94, 193-6. doi


2003 - PhD: Download
"Chemical Models of Zinc-dependent Enzymes: Methyltransferases and Class II Aldolases"
Department für Chemie und Biochemie, Universität Bern, Switzerland

1998 - Diploma:
"Simulação do Ciclo de Oxidação e Redução das Ubiquinonas num Modelo de Transporte Electrónico - Aplicação ao Peroxissoma"
Faculdade de Ciências da Universidade de Lisboa, Portugal

Software used in our Group

















AutoDock Vina



Computational Biochemistry: "Python Programing" (2nd Semester)

Molecular Simulation: "Molecular Mechanics/Molecular Dynamics" (2nd Semester)

Sistemas Químicos e Reactividade: "Computational Chemistry" (2nd Semester)


Miguel Ângelo dos Santos Machuqueiro

Invited Professor, Inorganic and Theoretical Chemistry Group, CQB, FCUL.

Email:  Phone: (+351) 217500112   Mobile: (+351) 967562285


Chemistry and Biochemistry Center, Faculty of Science, University of Lisbon
C8 Building (room 8.5.52), Campo Grande, 1749-016 Lisbon, Portugal

This page was created on Wednesday, 19-May-2010
...and it was last modified on Wednesday, 04-January-2017

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