Lab's Head

Miguel Machuqueiro is an Assistant Researcher at BioISI – BioSystems and Integrative Sciences Institute and the Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa (FCUL). He got his degree in Biochemistry from FCUL and a PhD in Bioorganic Chemistry from the University of Bern, Switzerland.

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90 Segundos de Ciência

During his pos-doc at ITQB-UNL, he started his research about the pH and redox potential effects on the structure and dynamics of biomolecules. After the pos-doc, he moved back to FCUL as an independent researcher and started his actual research lines, with special emphasis on the influence of pH on biomolecules at the water/membrane interface.

May 19, 2022

Telmo Silva (MSc.)

J. Ricardo Dias (MSc.)

Diogo Reis (MSc.)

Pedro Lopes (PosDoc)

Pedro Magalhães (PosDoc)

Diogo Vila Viçosa (MSc., PhD, and PosDoc)

Bruno Victor (PosDoc)

Vitor Teixeira (PosDoc)

Rafael Nunes (MSc.)

Bruno Calçada (MSc.)

Tomás J. Silva (MSc.)

Hugo Santos (MSc.)

Ana Sofia Capacho (MSc.)

João Henriques (MSc.)


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

In computational studies, the pH effects on biologically relevant molecules have been addressed with several limitations. Biological membranes are inherently complex and some important aspects, like protonation equilibrium, have also been unsatisfactorily modeled. The complexity of the membrane/water interface in biological membranes can even increase many-fold due to the presence of a myriad of different lipid molecules in their composition (e.g. anionic lipids). The convoluted contribution of the complex electrostatic interactions has rendered the problem of peptide and/or lipid protonation rather inaccessible, consequently deterring their study. However, the gradual raise of computational power, and the appearance of new and more refined force-fields, have made possible to model lipid bilayers patches with increased realism.

The so-called constant-pH MD (CpHMD) methods aimed at solving the problem of including the pH effects in MD simulations by allowing protonable groups to periodically change their state during the simulation, thereby capturing the coupling between conformation and protonation. In our group, we are actively developing new CpHMD strategies that enable us to study the dynamic properties of proteins and lipid bilayers under different conditions of temperature, pH, ionic strength, redox potential and solvent composition.

The pH-dependent membrane stability of pHLIP peptide studied using CpHMD simulations

The pH (Low) Insertion Peptide (pHLIP) is a 36 amino acid peptide derived from bacteriorhodopsin that targets tissues with acidic pH. It simultaneously targets tumors, carries the cargo, and translocate it across the plasma membrane at low pH values. At neutral pH, pHLIP is soluble as a monomer in water and associates with lipid bilayer surfaces largely as an unstructured peptide. Under acidic conditions, pHLIP inserts across a lipid bilayer with an apparent pK of 6, forming a transmembrane helix. The pH-dependent insertion process is coupled to the protonation of one or both of the Asp residues located in the transmembrane region of the peptide. We are studying the pH-dependent mechanism of action of pHLIP, taking advantage of the recent advancements in the constant-pH MD method for lipid bilayers (CpHMD-L).

Coupling of constant-pH MD methods with enhanced sampling techniques

We are developing new extensions to the stochastic titration method to allow it to be used within Replica-Exchange and Umbrella Sampling schemes simulations. This will allow us to circumvent several sampling limitations and calculate how protonation events influence the energy barriers associated with several phenomena, like lipid insertion or drug binding. These new extensions to the stochastic titration CpHMD method will improve significantly the conformation/protonation sampling in our simulations and allow us to tackle larger and even more exciting systems.

Including pH effects in membrane simulations

The lipid bilayer is the basic structural component of biological membranes. 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 micro-domain formation. We have extended to constant-pH MD methodology to allow for lipid protonation (CpHMD-L). This significantly increases the realism of our computational biophysics simulations and opens new opportunities to study phenomena happening at the membrane/water interface with an unprecedented level of detail.

New strategies to include pH effects at the water/membrane interface

Several peptides and proteins are able to insert into a biological membrane, even containing in their sequences amino acid residues that are usually charged at physiological pH. These molecules are able to shift their pKa values in favour of their neutral forms when interacting with lipid bilayers. The special environment created at the membrane/water interface makes it difficult to predict which protonation species is the most abundant at a given moment. We have developed a new approach, based on the CpHMD-L method) to follow the proton binding affinity of titratable groups along the membrane normal.

Simple compounds can modulate the permeability of lipid bilayers

Some compounds are able to cross lipid bilayers, others accumulate at the water/lipid interface and a very special class can even disrupt the membrane (e.g. by forming pores). Molecular dynamics simulations are an excellent complement to the most commonly used biophysical experimental techniques. From MD simulations, we can get molecular details on the interactions between our compounds and the bilayer, and even assess the perturbation created on the membrane by following some structural properties like membrane thickness, tail ordering or membrane permeability to model compounds. We collaborate with several experimentalist groups, providing them this information at the molecular level.

Protonation events can modulate protein/protein and drug/protein interactions

Most protein/protein interaction studies are done at constant protonation, even though a simple protonation event could change significantly the energetics of the whole process. Analogously, protein/drug binding affinities, which are usually measured in silico using molecular docking techniques, will depend on the protonation states of the residues in the active site pocket, and sometimes on the drug itself. We are using CpHMD methodologies to investigate how protonation influences the binding process of donepezil to acetylcholinesterase.

The role of electrostatics in the mechanism of action of KatG (Myc. tuberculosis)

Tuberculosis still ranks as the second leading cause of death from a single infectious agent, the Mycobacterium tuberculosis (Mtb) bacillus. Isoniazid (INH) is still the most effective drug against TB and remains the treatment of choice for tuberculosis. Several new INH derivatives have been tested and some exhibit dazzlingly good results. We have observed that the electrostatic environment around the heme pocket might be influencing KatG’s activity towards different INH derivatives. Therefore, we are investigating the correlation between some mutations around the pocket and the pKa values of key groups in the mechanism.

Study the pH-dependent conformational ensembles of highly-branched molecules

Dendrimers are very interesting molecules because of their unique properties as well as their potential applications. Many of these branched structures (PAMAM, PEI, PPI or peptide dendrimers) have titrable groups which render them sensitive to pH changes in solution. Because these molecules have high conformational freedom, their molecular structure has not yet been determined by experimental techniques, like X-ray crystallography. Therefore, we are using CpHMD simulations to study the conformational space of such molecules at different pH values and understand what factors define their structure in solution.


Projects We Lead
  • 2022 – 2023A fast deep learning approach to improve protein pKa predictions

    HPC: 840k CPU.core.hours + 4.5k GPU.hours :: 2021.09635.CPCA
  • 2015 – 2019CpHMD-L simulations of pHLIP peptides: design of new tumor-targeted drug delivery systems

    185k€ :: PTDC/QEQ-COM/5904/2014
  • 2011 – 2014Adding realism to the molecular modeling of lipidic membranes: inclusion of pH effects

    150k€ :: PTDC/QUI-BIQ/113721/2009
Projects We are Co-Responsible
  • 2018-2022Physical Basis of Disease: The case of Dialysis Related Amyloidosis

    195k€ :: PTDC/FIS-OUT/28210/2017
  • 2018-2022Targeting multi-resistant tuberculosis with new potent isoniazid derivatives: an integrated medicinal chemistry approach

    226k€ :: PTDC/MED-QUI/29036/2017
  • 2018-2022Deal with PAINS: strategies to identify membrane modulators

    235k€ :: PTDC/BIA-BFS/28419/2017
Projects we Participate In
  • Establishing a Pan-European Network on Computational Redesign of Enzymes - COZYME

    COST Action CA21162: Management Committee (2022)

  • TWIN2PIPSA - Twinning for Excellence in Biophysics of Protein Interactions and Self-Assembly


  • Ruthenium-peptide conjugates: arrows for selectively targeting breast cancer


  • Understanding and exploiting the impacts of low pH on micro-organisms - EuroMicropH

    CMST COST Action CA18113 (2019)

  • New diagnostic and therapeutic tools against multidrug resistant tumors - Stratagem

    CMST COST Action CM17104 (2018)

  • Uncovering blind spots in halogen bonding applications


  • Multivalent Glycosystems for Nanoscience - MultiGlycoNano

    CMST COST Action CM1102: Management Committee (2012)

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


  • Complexos de metais de transição usados como intercaladores de DNA: investigação teórica

    Bilateral Program: PESSOA (2011)

  • Síntese, estudos computacionais e propriedades biológicas de compostos de ouro

    Bilateral Program: FCT-CSIC (2010)

  • 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






A stochastic titration constant-pH molecular dynamics implementation for GROMACS. Currently under-development.


A python-based application to perform pKa calculations on peptides, proteins or lipid bilayers.


GROMOS compatible Molecular Mechanics Poisson-Boltzmann Surface Area calculations in protein-protein and protein-ligand systems.


A Protein Data Bank extension database of over 10M pKa and pI theoretical values.


A python wrapper for the Poisson-Boltzmann Equation solver DelPhi.


A fast and interpretable deep learning approach to accurate electrostatics-driven pKa prediction.


A python-based application to calculate membrane insertion and thickness from PDB trajectories.

pKa Profiler

A python script for pKa calculations along an interaction coordinate.