Protein foldingProtein folding

Tibor PáliInstitute of Biophysics

October 17, 2018

Tibor PáliInstitute of Biophysics

October 17, 2018

„Practice-oriented, student-friendly modernisation of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

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Evolution, genes and structure of proteinsEvolution, genes and structure of proteins

Given the rate at which proteins can change their conformation, the whole duration of evolution was not long enough to “try” all conformations, even for relatively small proteins. Protein folding is not a trial and error process!

Evolution and genes deal with codes (DNA and amino acid sequences) and functioning but not with structures.

Since amino acids have different physicochemical properties the sequence must be the primary determinant of the functional 3-dimensional structure.

There are cellular folding assistants, e.g. ribosome, chaperons, but they are “non-specific” since they merely accelerate the folding process of many proteins.

Therefore, the functional 3-dimensional structure of a protein is somehow fully determined (encoded) in its own sequence.

The encoding of structure can not be as precise as atomic resolution.

Since evolution developed genes to ensure biological functions, what is encoded in the sequence of a protein is its functional fold in its native environment at a reduced resolution (probably residue resolution).

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Wikipedia Commons

Hierarchy of protein structuresHierarchy of protein structures

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Wikipedia Commons

Un-folded (denatured, non-functional)Un-folded (denatured, non-functional) Folded (native, functional)Folded (native, functional)

Basic structural forms (folds) of a proteinBasic structural forms (folds) of a protein

The reverse process is unfolding or denaturing. Neither of these forms (states) are necessarily static and unique.There can be, and usually are, intermediate forms (states) too.There can be, and usually are, different native folds during functioning.

FoldingFolding

Protein folding is the process of forming a native, functional protein from a denatured, non-functional state.

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Wikipedia Commons

Illustration of alternative folding pathwaysIllustration of alternative folding pathways

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Isolated systems under constant pressure go in a direction which decreases the Gibbs free energy (G).

A change in G during a process, such as folding, has enthalpy and entropy contributions:

∆G = ∆H - T * ∆S,

where ∆H and ∆S is the change in enthalpy and entropy, respectively, during the process at temperature T.

Enthalpy is related to the internal energy of the system, entropy is related to the number of possible states at a given internal energy.

Protein folding (and unfolding) involves rearrangement of residue-residue and residue-solvent interactions, which affect both enthalpy and entropy.

Unfolding becomes irreversible if the unfolded protein forms oligomers with much lower G than that of the native, folded state.

Thermodynamics of protein foldingThermodynamics of protein folding

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Wikipedia Commons

The main driving force is the hydrophobic effect, which minimises unfavourable hydrophobic-hydrophilic interactions mainly via rearrangement of H-bonds.

The hydrophobic effect is entropy-driven: interfacial water molecules around an apolar solute form a cavity that is energetically preferable to pure water but entropically is much less preferable.

There are also contributions from changes in other residue-residue interactions, e.g. S-S bridges, metal complexes, structural co-factors in the proteins.

Water-soluble proteinsWater-soluble proteins

Hydrophobic aaHydrophobic aa

Hydrophilic aaHydrophilic aa

∆G∆G

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Membrane proteins— at the forefront of biophysical research

Membrane proteins— at the forefront of biophysical research

The vacuolar proton pumping ATP-ase (V-ATPase)The vacuolar proton pumping ATP-ase (V-ATPase)

Membrane Biophysics Group, based on data published inFerencz, C., Petrovszki, P., Kota, Z., Fodor-Ayaydin, E., Haracska, L., Bota, A., Varga, Z., Der, A., Marsh, D. and Pali, T. (2013) Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes. Eur. Biophys. J. 42, 147–158.

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Hydrophobic matchingand trans-membrane proteins

Hydrophobic matchingand trans-membrane proteins

polar (hydrophilic)polar (hydrophilic)apolar (hydrophobic)apolar (hydrophobic)

polar (hydrophilic)polar (hydrophilic)

polar (hydrophilic)polar (hydrophilic)

apolar (hydrophobic)apolar (hydrophobic)

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The two main classesof trans-membrane proteins

The two main classesof trans-membrane proteins

Alpha-helix bundlesAlpha-helix bundles Beta-barrelsBeta-barrels

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Membrane proteins are most challengingMembrane proteins are most challenging

The lipid bilayer is an anisotropic and inhomogeneous solvent, which reduces the conformational space of membrane proteins.

However, the lipid bilayer provides one hydrophobic and two hydrophilic layers in addition to the aqueous phase to proteins. This system is therefore much more complex than that of water soluble proteins.

Because of the much more complex environment, membrane proteins are much less accessible to classical structural biology techniques (X-ray diffraction and NMR spectroscopy) for structure determination than water soluble proteins. (A glance at the PDB database demonstrates this statement very clearly.)

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Structure determination of membrane proteinsStructure determination of membrane proteins

Extracting a membrane protein from its native lipid environment is not only difficult but it also may alter its conformation and make it non-functional. Functionally relevant structural data, even if low resolution, are very important for structure determination or prediction of membrane proteins.

Crystallisedin a folded form (for X-ray).

Solubilised in a folded form(for, e.g., NMR).

Solubilised in an unfolded form (for folding studies).

Structure determination of membrane proteins is much behind that of water-soluble proteins. The main reason is that substitution of the lipid environment with other molecules, that should aid solubilisation or crystallisation, is a very challenging task and there are no uniform recipes (mainly a trial and error process).

Membrane protein in its native environmentMembrane protein in its native environment

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Thermodynamics and kineticsof membrane protein foldingThermodynamics and kineticsof membrane protein folding

Each step, represented with arrows, involves a change in Gibbs free energy (G). The larger the change (∆G), the faster the rate of transition (in the direction of decreasing G).

Based on data published in

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Cytochrome c oxidase (dimer)

Some membrane proteins have lipidsas structural co-factors

Some membrane proteins have lipidsas structural co-factors

Membrane Biophysics Group, based on data published inMarsh, D. and Pali, T. (2013) Orientation and conformation of lipids in crystals of transmembrane proteins. European Biophysics Journal 42, 119–146; andMarsh, D. and Pali, T. (2006) Lipid conformation in crystalline bilayers and in crystals of transmembrane proteins. Chemistry and Physics of Lipids 141, 48-65.

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Common techniques to measure protein foldingCommon techniques to measure protein folding

Differential scanning calorimetry.

Fourier-transform infrared spectroscopy.

Circular dichroism optical absorption spectroscopy.

Fluorescence spectroscopy.

Site-directed spin-label electron paramagnetic resonance spectroscopy.

X-ray diffraction.

Nuclear magnetic resonance spectroscopy.

Surface plasmon resonance.

(The list is not complete. Any technique measuring protein structure, dynamics, thermodynamics and kinetics can be used.)

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Note: The practical demonstration will be on DSC and FTIR for measuring thermodynamic and structural characteristics of the thermal unfolding of Lysozyme.

Differential scanning calorimetry (DSC)Differential scanning calorimetry (DSC)

Fourier-transform infrared spectroscopy (FTIR)Fourier-transform infrared spectroscopy (FTIR)

DSC is used to determine the heat capacity (Cp = q/∆T) of a protein as a function of temperature. It allows studying the thermodynamics of folding and unfolding of the protein.FITR is used to detect molecular vibrations. It can be used to determine the secondary structure of a protein because the amide vibrations (of the protein backbone) are sensitive to it. A combination of polarised excitation and attenuated total reflection can be used to determine the orientation of secondary structure segments of membrane proteins in a membrane.

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Polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy

Lipid (CH stretching)

Protein(Amide I-II)

Structure, dynamics

Lipid (CO)Lipid (CO)

D2O

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Fluorescence spectroscopyFluorescence spectroscopy

Membrane Biophysics Group, unpublished

This technique can be used to detect intrinsic fluorescence from fluorescent (aromatic) amino acids (tryptophan, tyrosine, phenylalanine) or that of synthetic dyes attached to desired amino acids. Various spectral parameters depend on protein structure.

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Protein-lipid interactionProtein-lipid interaction

Lipids restricted in dynamics by a membrane protein detected by spin label electron paramagnetic (EPR) spectroscopy can be used to measure the intra-membranous perimeter of the proteins, as well as specific protein-lipid interactions.

Membrane Biophysics Group, based on data published inPali, T. and Kota, Z. (2013) Studying lipid-protein interactions with electron paramagnetic resonance spectroscopy of spin-labeled lipids. In: Lipid-Protein Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 974, Jorg H. Kleinschmidt (ed.), Springer New York 2013, pp. 297-328.

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Theoretical approaches

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Example: an experimental approach to the thermal unfolding of lysozymeExample: an experimental approach to the thermal unfolding of lysozyme

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Membrane Biophysics Group, unpublished

Denaturation of Lysozyme in solution (DSC)Denaturation of Lysozyme in solution (DSC)

There are two transitions detected (labeled as “L” and “H”).

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3 3.5 4 4.5 7.4pH

79% 81% 82% 79% 0%

Membrane Biophysics Group, unpublished

Denaturation of Lysozyme in solution: DSC, reversibility, effect of pHDenaturation of Lysozyme in solution: DSC, reversibility, effect of pH

Denaturation temperatureDenaturation temperature Reversibility (%)Reversibility (%)

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LysozymeL/P=5.3 (mol/mol)L/P=16 (mol/mol)

L/P=5.3 sym.-CH2

L/P: lipid-protein molar ratio

Membrane Biophysics Group, unpublished

Denaturation of Lysozyme in solution:FTIR, secondary structure, lipid-protein interactionDenaturation of Lysozyme in solution:FTIR, secondary structure, lipid-protein interaction

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Membrane Biophysics Group, unpublished

∆G∆G “L” transition“L” transition

“H” transition“H” transition“I”, irreversible aggregation“I”, irreversible aggregation

The model of the thermal unfolding of LysozymeThe model of the thermal unfolding of Lysozyme

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Membrane Biophysics Group, based on data published in

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Condition (1):

His 20 mM, no preincubation

Kidney ATPase Shark ATPase

Membrane Biophysics Group, based on data published in

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Membrane Biophysics Group, based on data published inKota, Z., Pali, T. and Marsh, D. (2004) Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized ATR infrared spectroscopy and spin-label electron spin resonance. Biophysical Journal 86(3), 1521-1531.

The alternative dimeric forms of gramicidin A (antibiotic polypeptide) in a membraneThe alternative dimeric forms of gramicidin A (antibiotic polypeptide) in a membrane

31Membrane Biophysics Group, based on data published in

32Membrane Biophysics Group, based on data published in

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Example: a theoretical approach to predict the basic fold of cytochrome b561 familyExample: a theoretical approach to predict the basic fold of cytochrome b561 family

Utilising bibliographic data on structure and function.

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Utilising bibliographic data on structure and functionfor structure predictions

Utilising bibliographic data on structure and functionfor structure predictions

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Sequence analysis for predicting conserved trans-membranehelices and their orientation

Sequence analysis for predicting conserved trans-membranehelices and their orientation

Membrane Biophysics Group, based on data published inBashtovyy, D., Berczi, A., Asard, H. and Pali, T. (2003) Structure prediction for the di-heme cytochrome b-561 protein family. Protoplasma 221(1-2), 31-40.

Pilpel’s kPROT method (Pilpel Y, Ben-Tal N, Lancet D (1999) kPROT: a knowledge-based scale for the propensity of residue orientation in transmembrane segments. Application to membrane protein structure prediction. J. Mol. Biol. 294: 921–935).

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Membrane Biophysics Group, based on data published inBashtovyy, D., Berczi, A., Asard, H. and Pali, T. (2003) Structure prediction for the di-heme cytochrome b-561 protein family. Protoplasma 221(1-2), 31-40.

Predicted basic foldof a trans-membrane electron transporter family

Predicted basic foldof a trans-membrane electron transporter family

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-oriented, student-friendly modernisation of the biomedical

education for strengthening the international competitiveness of the rural Hungarian universities "

TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.

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Thank you for your attention!Thank you for your attention!