Melinda Noronha website | Instituto Superior Técnico

Melinda Noronha (PhD)	Departamento de Engenharia Química e Biológica (DEQB) Instituto Superior Técnico Universidade Técnica de Lisboa Av. Rovisco Pais - 1049-001 Lisboa Portugal telefone: (+351) 21 8419606 email: mnoronha@ist.utl.pt
Scientific Research		Publications
My areas of research include: 1. Protein fluorescence 2. Mechanisms of protein stabilization by osmolytes Protein fluorescence My Ph.D. was focused on the application of picosecond time-resolved fluorescence spectroscopy (TRFS) to discriminate the fluorescence signal of proteins with only tyrosine residues namely, ubiquitin (1 Tyr), cytochrome c’’ (2 Tyr) and Ribonuclease A (6 Tyr). Upon discrimination, the fluorescence signal of individual tyrosine residues was used to track local conformational changes resulting from thermal and chemical unfolding in a protein with multiple chromophores. The three proteins studied also served as model case studies for the presence of three principal quenching mechanisms possible in proteins, namely, electron transfer (from tyrosine residues to disulfide bridges as seen in Ribonuclease A), proton transfer (from a single tyrosine residue to a nearby carboxylate group of a glutamic acid in ubiquitin) and finally energy transfer (from the tyrosine residue to the haem of cytochrome c’’) and how these mechanisms affect the fluorescence decay times of tyrosine residues. Mechanisms of protein stabilization by osmolytes The use of compatible solutes (sugars or polyols accumulated as a response to stress) to stabilize proteins is a strategy adopted by the pharmaceutical, medical and food industries and therefore of commercial importance. However, the molecular mechanism as to how exactly these compounds stabilize proteins remains unknown. It is generally accepted that most compatible solutes or osmolytes are excluded from the surface of the protein and the stabilizing effect arises not as a direct interaction of the solute with the protein but because energetically the compatible solute destabilizes the unfolded state and therefore shifts the equilibrium towards the native state of the protein. Thermodynamic studies carried out by Faria et al. have shown that the stabilizing effect of a hypersolute, mannosylglycerate, results because of an entropic gain. Also, an increase in the ∆Cp was observed from 2 to 4 kcal K-1 mol-1 in the absence and presence of the 0.5 M mannosylglycerate. Our objective is to further our understanding by carrying out kinetics of folding/unfolding in the presence of solutes and see if the rate of folding or the rate of unfolding or both are affected. Initial work on protein folding kinetics and protein stabilization, using the ns-LASER-T-jump technique at the University of Leeds (UK), has shown that the model proteins bovine ubiquitin (UBQ) and Staphylcoccus aureus nuclease A (Snase) do not unfold in the s-2 ms time range. In 2007, a new T-jump accessory was acquired and coupled to an existing conventional stopped-flow (BioLogic), which allows us to measure the kinetics of thermal folding/unfolding of any protein from 4 ms and above. The nature of the decay functions (single or multiple exponential) observed will provide primary detailed information on the simplicity or complexity of the unfolding mechanism. Research Projects 1. Molecular basis for protein thermostabilisation by hypersolutes POCTI/3513/BME/2000 (member of team) 2. Mechanism and Kinetics of Protein Stabilisation by Osmolytes POCI/QUI/56585/04 (member of team) 3. Structural determinants of protein stabilization by compatible solutes from hyperthermophiles: in search of guidelines solute improvement POCI/BIA-PRO/57263/2004 (member of team)	Papers in international scientific periodicals with referees 1. Petersen, M.T.N, Petersen, E., Fojan, P., Noronha, M., Madsen, R.G and Petersen, S.B. 2001. "Engineering the pH-optimum of a triglyceride lipase: from Predictions Based on Electrostatic Computations to Experimental Results", J. Biotechnology, 87, 225-254. 2. Noronha, M., Lima, J.C., Lamosa, P., Santos, H., Maycock, C., Ventura, R., Maçanita, A.L. 2004 “Intramolecular fluorescence quenching of tyrosine by the peptide α-carbonyl group revisited”, J. Phys. Chem. A., 108, 2155-2166. 3. Noronha, M.; Lima, J.C.; Bastos, M.; Santos, H.; Maçanita, A.L. 2004. Biophys. J. “Unfolding of ubiquitin studied by picosecond time-resolved fluorescence of the tyrosine residue”, 87: 2609-2620. 4. Noronha, M.; Lima, J.C.; Paci, E; Santos, H.; Maçanita, A.L. 2007. Biophys. J. “Tracking local conformational changes of ribonuclease A using picosecond time-resolved fluorescence of the six tyrosine residues”, 92: 4401-4414. Papers in conference proceedings 1. Noronha, M.; Lima, J.C.; Santos, H.; Maçanita, A.L. 2005. FEBS Journal. “The discrimination of tyrosine residues in ribonuclease A by picosecond time-resolved fluorescence spectroscopy,” 272: 382-382 Suppl. 1.

Melinda Noronha

melinda

(PhD)

Departamento de Engenharia Química e Biológica (DEQB)
Instituto Superior Técnico
Universidade Técnica de Lisboa
Av. Rovisco Pais - 1049-001
Lisboa
Portugal

telefone: (+351) 21 8419606
email: mnoronha@ist.utl.pt

Scientific Research

Publications

My areas of research include:
1. Protein fluorescence
2. Mechanisms of protein stabilization by osmolytes

Protein fluorescence
My Ph.D. was focused on the application of picosecond time-resolved fluorescence spectroscopy (TRFS) to discriminate the fluorescence signal of proteins with only tyrosine residues namely, ubiquitin (1 Tyr), cytochrome c’’ (2 Tyr) and Ribonuclease A (6 Tyr). Upon discrimination, the fluorescence signal of individual tyrosine residues was used to track local conformational changes resulting from thermal and chemical unfolding in a protein with multiple chromophores. The three proteins studied also served as model case studies for the presence of three principal quenching mechanisms possible in proteins, namely, electron transfer (from tyrosine residues to disulfide bridges as seen in Ribonuclease A), proton transfer (from a single tyrosine residue to a nearby carboxylate group of a glutamic acid in ubiquitin) and finally energy transfer (from the tyrosine residue to the haem of cytochrome c’’) and how these mechanisms affect the fluorescence decay times of tyrosine residues.

Mechanisms of protein stabilization by osmolytes
The use of compatible solutes (sugars or polyols accumulated as a response to stress) to stabilize proteins is a strategy adopted by the pharmaceutical, medical and food industries and therefore of commercial importance. However, the molecular mechanism as to how exactly these compounds stabilize proteins remains unknown. It is generally accepted that most compatible solutes or osmolytes are excluded from the surface of the protein and the stabilizing effect arises not as a direct interaction of the solute with the protein but because energetically the compatible solute destabilizes the unfolded state and therefore shifts the equilibrium towards the native state of the protein. Thermodynamic studies carried out by Faria et al. have shown that the stabilizing effect of a hypersolute, mannosylglycerate, results because of an entropic gain. Also, an increase in the ∆Cp was observed from 2 to 4 kcal K-1 mol-1 in the absence and presence of the 0.5 M mannosylglycerate. Our objective is to further our understanding by carrying out kinetics of folding/unfolding in the presence of solutes and see if the rate of folding or the rate of unfolding or both are affected. Initial work on protein folding kinetics and protein stabilization, using the ns-LASER-T-jump technique at the University of Leeds (UK), has shown that the model proteins bovine ubiquitin (UBQ) and Staphylcoccus aureus nuclease A (Snase) do not unfold in the s-2 ms time range. In 2007, a new T-jump accessory was acquired and coupled to an existing conventional stopped-flow (BioLogic), which allows us to measure the kinetics of thermal folding/unfolding of any protein from 4 ms and above. The nature of the decay functions (single or multiple exponential) observed will provide primary detailed information on the simplicity or complexity of the unfolding mechanism.

Research Projects
1. Molecular basis for protein thermostabilisation by hypersolutes POCTI/3513/BME/2000 (member of team)
2. Mechanism and Kinetics of Protein Stabilisation by Osmolytes POCI/QUI/56585/04 (member of team)
3. Structural determinants of protein stabilization by compatible solutes from hyperthermophiles: in search of guidelines solute improvement POCI/BIA-PRO/57263/2004 (member of team)

Papers in international scientific periodicals with referees
1. Petersen, M.T.N, Petersen, E., Fojan, P., Noronha, M., Madsen, R.G and Petersen, S.B. 2001. "Engineering the pH-optimum of a triglyceride lipase: from Predictions Based on Electrostatic Computations to Experimental Results", J. Biotechnology, 87, 225-254.

2. Noronha, M., Lima, J.C., Lamosa, P., Santos, H., Maycock, C., Ventura, R., Maçanita, A.L. 2004 “Intramolecular fluorescence quenching of tyrosine by the peptide α-carbonyl group revisited”, J. Phys. Chem. A., 108, 2155-2166.

3. Noronha, M.; Lima, J.C.; Bastos, M.; Santos, H.; Maçanita, A.L. 2004. Biophys. J. “Unfolding of ubiquitin studied by picosecond time-resolved fluorescence of the tyrosine residue”, 87: 2609-2620.

4. Noronha, M.; Lima, J.C.; Paci, E; Santos, H.; Maçanita, A.L. 2007. Biophys. J. “Tracking local conformational changes of ribonuclease A using picosecond time-resolved fluorescence of the six tyrosine residues”, 92: 4401-4414.

Papers in conference proceedings
1. Noronha, M.; Lima, J.C.; Santos, H.; Maçanita, A.L. 2005. FEBS Journal. “The discrimination of tyrosine residues in ribonuclease A by picosecond time-resolved fluorescence spectroscopy,” 272: 382-382 Suppl. 1.