Protein Stability and Electrostatic Interactions - PowerPoint PPT Presentation

1 / 31
About This Presentation
Title:

Protein Stability and Electrostatic Interactions

Description:

Laboratory of Experimental and Computational Biology, NCI Frederick, Frederick, Maryland, USA. ... (DsCs) as compared to chicken citrate synthase (GgCs) ... – PowerPoint PPT presentation

Number of Views:186
Avg rating:3.0/5.0
Slides: 32
Provided by: ruthnu
Category:

less

Transcript and Presenter's Notes

Title: Protein Stability and Electrostatic Interactions


1
Protein Stability and Electrostatic Interactions
  • Sandeep Kumar, Ph.D.
  • Laboratory of Experimental and Computational
    Biology, NCI Frederick,
  • Frederick, Maryland, USA.
  • URL www.lecb.ncifcrf.gov/kumarsan
  • Email kumarsan_at_ncifcrf.gov

2
Research projects
  • Protein thermodynamic data analysis and modeling.
  • Molecular adaptations by extremophilic proteins.
  • Contribution of electrostatic interactions
    towards protein stability.
  • Hierarchical nature of protein folding and
    binding.
  • Coupling between protein folding and function.
  • Structural disorder and protein misfolding.
  • Implications of energy landscape theory for
    folding and binding of 'real' proteins.

3
Talk content
  • Protein folding and stability.
  • Description of forces that act in protein
    structure.
  • Elementary protein thermodynamics and protein
    stability curve.
  • Thermodynamic comparison of thermophilic and
    mesophilic proteins and formation of additional
    specific interactions in the thermophilic
    proteins.
  • Structural and sequence comparison of
    thermophilic and mesophilic proteins.
  • Electrostatic interactions and protein stability.
  • Fluctuation in ion pairs and their stabilities in
    proteins.
  • Relationship between ion pair geometry and
    stability.
  • Conclusions.
  • Postscript.
  • Acknowledgements.

4
Protein folding and stability
  • Amino acid sequence of a protein codes for
    protein three dimensional structure and function.
  • It also codes for protein folding kinetics and
    presence / absence of stable intermediates.
    Sequential and non-sequential folding.
  • Stability of the native protein fold and its
    response to the changes in proteins environment,
    e. g., temperature, pH, salt, solvent,
    presence/absence of proteins substrates, ligands
    or denaturants (Urea, GdmHCl).
  • Localization in the cell.

5
Forces that act in protein structure
  • A folded protein is quite like Gulliver tied down
    by Lilliputians. Except that they act from within
    and keep the protein active.
  • There are two kinds of interactions in proteins,
    specific and non-specific.
  • Non-specific interactions are largely hydrophobic
    and arise due to burial of apolar residues in the
    protein structure.
  • Specific interactions are largely electrostatic
    interactions, such as hydrogen bonds, salt
    bridges or ion pairs.

6
Elementary protein thermodynamics
  • Thermodynamics of protein folding and unfolding
    can be studied via thermal and chemical
    denaturations using experimental techniques such
    as UV/VIS, Fl, CD, DSC and ITC.
  • Simple proteins show reversible two state N?D
    transitions.
  • The variation in proteins stability as a
    function of temperature yields protein stability
    curve described by Gibbs-Helmoholtz equation
  • ?G(T) ?HG (1-T/TG) - ?Cp (TG - T) T
    ln(T/TG)
  • The parameters TG, ?HG and ?Cp are determined
    using experimental procedures.

7
  • A protein stability curve yields several cardinal
    characteristics of the protein
  • Slope at TG -?HG / TG - ?SG
  • Curvature at TG -?Cp / TG
  • Temperature of maximal protein stability,
  • TS TG exp (-?HG / TG ?Cp)
  • Maximal protein stability,
  • ?G(TS) ?HG - (TG TS) ?Cp
  • Most two-state proteins with large enough
    hydrophobic cores are maximally stable around the
    room temperature.
  • S. Kumar, C. J. Tsai and R. Nussinov, 2002,
    Biochemistry, 41, 5359-5374.

8
Thermodynamic comparison of thermophilic and
mesophilic proteins
  • We have compared the thermodynamic features of
    homologous thermophilic and mesophilic proteins.
  • In literature, such data is available for five
    families of homologous thermophilic and
    mesophilic proteins. All the proteins in these
    families show reversible two state N?D
    transition. These families contain 19 proteins.
  • Two families containing 11 mesophilic proteins
    were used as control.
  • S. Kumar, C. J. Tsai and R. Nussinov, 2001,
    Biochemistry, 40, 14152-14165.

9
  • Our results show that protein stability curves of
    thermophiles are upshifted and broadened.

10
  • Melting temperature is correlated with maximal
    protein stability, enthalpy and entropy changes
    at melting temperature. This indicates that
    protein thermostability involves formation of
    additional specific interactions.

11
Sequence and structural comparison of homologous
thermophilic and mesophilic proteins
  • All sequence/structural parameters in 18
    non-redundant families of homologous thermophilic
    and mesophilic proteins have been compared.
  • Structural properties that define the protein
    fold, such as hydrophobicity, atomic packing,
    main chain hydrogen bonds remain constant.
  • Thermophilic proteins prefer residues with longer
    side chains, e. g. Arg and Tyr, and have greater
    ?-helical content.
  • A majority of the thermophilic proteins are have
    greater number of salt bridges and their networks
    both within subunits and across the subunit
    interfaces.
  • S. Kumar, C. J. Tsai and R. Nussinov, 2000,
    Protein Engineering, 3, 179-191.

12
Electrostatic interactions and Protein stability
  • Biochemical intuition tells us that electrostatic
    interaction between the oppositely charged
    residues would be favorable. But, salt bridge and
    ion pair formation can be destabilizing in
    proteins.
  • Transfer of a salt bridge from water to nonpolar
    environment costs 10 - 16 kcal/mol (B. Honig and
    W. L. Hubell, 1984, PNAS 81, 5412-5416).
  • The energy penalty paid due to the desolvation of
    the charged residues may not be recovered by
    favorable interaction among the charged residues.
    In fact, a previous study had shown that most of
    the salt bridges are destabilizing towards
    proteins (Z. Hendsch and B. Tidor, 1994, Protein
    Science, 3, 211-226).

13
  • Arc repressor gained in stability upon mutation
    of the buried charged residues, that form a salt
    bridge triad, in its core with the hydrophobic
    residues (Waldburger et al., 1995, Nature Struct.
    Biol. 2, 122-128).
  • Other investigators had found the salt bridges to
    be stabilizing (e.g. S. Marqusee and R. Sauer,
    1994, Protein Science 3, 2217-2225 D. Xu, C. J.
    Tsai, R. Nussinov, 1997, JMB 265, 68 - 84).
  • These observations indicate that there is scope
    to further understand the fundamental nature of
    electrostatic interactions in proteins.
  • If most salt bridges are destabilizing, then why
    should they occur with greater frequency in the
    thermophilic proteins?

14
Continuum electrostatics calculations
  • In classical electrostatics, a homogeneous medium
    has a dielectric constant that measures its bulk
    polarizability. Hence, the medium can be
    considered as a "continuum" and polarization of
    atoms is not treated explicitly. In such
    situations, coulomb's law can fully describe the
    interaction between any two charges, qi and qj.
  • The situation in proteins is more complicated.
    Proteins have usually non-homogenous charge
    distributions and have low dielectric constants
    (?) At the same time, the solvent water has high
    dielectric constant.

?80
.qi
.qj
? 4
15
  • Poisson-Boltzmann equation can model the
    electrostatic effects in proteins more
    accurately.
  • The continuum electrostatic calculations treat
    the protein in full atomic details but the
    solvent (water) is treated only in terms of its
    bulk properties . These calculations can be used
    to estimate the electrostatic free energy
    contribution of a salt bridge towards protein
    stability.
  • The electrostatic free energy contribution of a
    salt bridge is computed with respect to
    hydrophobic isosteres of the salt bridging
    charged residues. The hydrophobic isosteres are
    nothing but the salt bridging residue side chains
    with their partial atomic charges set to zero.

16
(No Transcript)
17
Salt bridges and their networks in Pyrococcus
furiosus Glutamate dehydrogenase
  • Pyrococcus furiosus Glutamate dehydrogenase
    (PfGDH) and Clostridium symbiosum gluatamate
    dehydrogenase (CsGDH) share 34 sequence
    identity. Their 3-D structures superimpose with
    RMSD of 1.38 Å.
  • PfGDH has TG of 113?C while CsGDH has TG of 55?C.
    The crystal structures of PfGDH (1GTM) indicates
    70 increase in salt bridges over that of CsGDH
    (1HRD).
  • Salt bridges in a monomer of PfGDH form extensive
    networks and cooperatively stabilize one another.
  • Our calculations show that salt bridges in PfGDH
    are highly stabilizing while those in CsGDH are
    only marginally stabilizing.
  • S. Kumar, B. Ma, C. J. Tsai and R. Nussinov,
    2000, Proteins, 38, 368-383.

18
Salt bridges in monomeric proteins
  • We have carried out a statistical survey of 222
    salt bridges in 36 non-homologous monomeric
    proteins with high resolution crystal structures
    (1.6 Å resolution or better).
  • Salt bridge formation is inferred for a pair of
    oppositely charged residues (Asp or Glu with Arg,
    Lys or His) if they meet the following criteria
    (i) The centroids of the side chain charged
    groups lie within 4 Å of each other and (ii)
    atleast a pair of Asp or Glu side chain carboxyl
    oxygen and Arg, Lys or His side chain nitrogen
    atoms are also within a distance of 4 Å.

19
Salt bridges in monomeric proteins
  • Most (86) of the salt bridges are stabilizing
    towards proteins, regardless of whether they are
    buried or exposed, isolated or networked,
    hydrogen bonded or non-hydrogen bonded.
  • A major finding of this work is that geometrical
    orientation of the side chain charged groups in
    the salt bridging residues is a critical factor
    in determining the salt bridge stability. Hence,
    salt bridges with favorable geometries are likely
    to be stabilizing anywhere in the protein
  • Majority of salt bridges are formed between the
    charged residues that are close in amino acid
    sequence also.
  • S. Kumar and R. Nussinov, 1999, J. Mol. Biol.
    293, 1241-1255.

20
Salt bridges in protein crystal and NMR structures
  • Usually protein crystal structures provide static
    pictures. However, proteins often show systemic
    and segmental flexibilities.
  • Segmental flexibility refers to motion of protein
    parts e. g. hinge bending. Across the moving
    parts of the proteins, the salt bridge formation
    is avoided.
  • N. Sinha, S. Kumar and R. Nussinov, 2001,
    Structure, 9, 1165 - 1181.
  • Systemic flexibility arises due to the motion of
    protein backbone and side chain atoms. This
    affects stability of the salt bridges. We have
    computed electrostatic strengths of ion pairs
    using NMR conformer ensembles of proteins.

21
Ion pairs in c-Myc-Max leucine zipper
  • c-Myc-Max leucine zipper has 4 inter-helical and
    2 intra-helical ion pairs and a five residue ion
    pair network (IPN-5).
  • Continuum electrostatic calculations reveal that
    these ion pairs fluctuate between being
    stabilizing and destabilizing in different NMR
    conformers.
  • These fluctuations are due to the variations in
    location of the ion pairing residues as well as
    geometrical orientation of the side chain charged
    groups in the ion pair.
  • S. Kumar and R. Nussinov, 2000, Proteins, 41,
    485-497.

22
(No Transcript)
23
Fluctuations in ion pairs and their stabilities
  • We have surveyed 22 ion pairs in 14 NMR conformer
    ensembles (with 40 conformers) of 11
    non-homologous monomeric proteins. These ion
    pairs form salt bridges in crystal structures,
    NMR average structures or most representative
    conformers of the proteins.
  • Most ion pairs show fluctuations and interconvert
    between being stabilizing and destabilizing. Salt
    bridges observed in the crystal structures easily
    break and reform in the NMR conformer ensembles.
  • Hence, formation of ion pairs and their
    stabilities are conformer population dependent.
  • S. Kumar and R. Nussinov, 2001, Proteins, 43,
    433-454.

24
Relationship between Ion pair geometry and
electrostatic strength
  • Ion pair geometry is defined by two parameters, r
    and ?.
  • r is the distance between the centroids of side
    chain charged groups in the ion pairing residues.
  • ? is the angle between two unit vectors. Each
    unit vector joins a Ca atom and a side chain
    charged group centroid in an ion pairing residue.
    We actually take the supplemental angle.

25
  • We have used the data on NMR conformer ensembles.
    We find it convenient to divide the ion pairs
    into three types, namely, salt bridges, N?O
    bridges and longer range ion pairs.
  • Most (92) of the salt bridges are stabilizing.
    For N?O bridges, the stabilizing proportion drops
    to (68). Two thirds (67) of the longer range
    ion pairs are destabilizing.
  • Salt bridges have the strongest electrostatic
    strengths. The electrostatic strengths of N?O
    bridges are considerably weaker than those of the
    salt bridges. Long range ion pairs are the
    weakest.
  • We find that most of the ion pairs are
    stabilizing if their side chain charged group
    centroids fall within 5 Å.
  • S. Kumar and R. Nussinov, 2002, Biophysical
    Journal, 83, 3, 1595 - 1612.

26
Conclusions
  • Close-range electrostatic interactions play
    important roles in protein stability and
    flexibility.
  • Increased formation of salt bridges and their
    networks is one of the most consistent factors
    that contribute towards the stability of the
    thermophilic proteins.
  • However, whether salt bridges would be
    stabilizing or destabilizing depends upon the
    geometrical orientation of the side chain charged
    groups.
  • Both the identity and of the residues forming the
    close-range electrostatic interactions, such as
    salt bridges and ion pair, as well as their
    electrostatic strengths fluctuate due to protein
    flexibility. Hence, formation of these
    interactions is conformer population dependent.
  • These observations have important implications
    for de novo protein design and rational
    manipulation of protein stability.

27
Postscript
  • Electrostatic destabilization also plays
    important roles in molecular adaptation at low
    temperatures as in case of psychrophilic Citrate
    synthase.
  • Pyrococcus furiosus Citrate synthase (PfCs) shows
    greater sequence and structural similarities with
    psychrophilic antarctic bacterium Ds2-3R citrate
    synthase (DsCs) as compared to chicken citrate
    synthase (GgCs). All three are homodimers.
  • Both PfCs and DsCs contain greater extents of
    charged residues, salt bridges and their networks
    as compared to GgCs. Salt bridges and their
    networks are stabilizing towards both PfCs and
    DsCs.
  • Where is the difference?

28
Different roles of protein electrostatics
  • Salt bridges in the hyperthermophilic citrate
    synthases are largely concentrated in the active
    site regions and dimer interface while they are
    dispersed throughout the structure in the
    psychrophilic citrate synthase.
  • The continuum electrostatic calculations suggest
    that the charged residues in active site regions
    of psychrophilic citrate synthase are highly
    destabilizing.
  • Physical properties of water, namely, viscosity,
    surface tension and dielectric constant are
    different at 0C and 100C.
  • The electrostatic free energy contributions by
    individual charged residues show greater
    fluctuations in the psychrophilic citrate
    synthase.

29
(No Transcript)
30
Interpretation
  • At high temperatures, the hyperthermophilic
    citrate synthase needs to guard against the loss
    of native structure, particularly in the active
    site region and dimer interface. The increased
    and stabilizing electrostatic interactions can
    resist disorder in these regions.
  • The charged residues and electrostatic
    interactions in the psychrophilic citrate
    synthase ensure proper hydration of the protein
    at low temperatures.
  • The higher catalytic effeciency of psychrophilic
    enzymes originates from their flexibility,
    particularly in the active site region. The
    highly destabilizing charged residues in the
    active site may contribute towards its greater
    flexibility.
  • These observations indicate that protein
    electrostatics may play important roles in both
    heat and cold adaptations by citrate synthase.
  • S. Kumar and R. Nussinov, 2002, submitted.

31
Acknowledgements
  • Profs. Ruth Nussinov and Jacob V. Maizel Jr. at
    LECB, NCI-Frederick, NIH, USA.
  • C. J. Tsai, Buyong Ma, Jeng Zian Hu, Dong Xu,
    Neeti Sinha, Yuk Yin Sham, K. Gunasekaran and
    David Zanuy.
  • Drs. Tom Schneider (NCI) and N. Pattabiraman
    (Georgetown Univ.).
  • Students at Tel Aviv University, esp. Adi
    Barzilai.
  • Prof. Manju Bansal, MBU, IISc., Bangalore, India.
  • D. Mohanty (NII), M. Ravikiran, B. Choudhary,
    Shibasis Choudhary, R. Velavan and Anirban Ghosh.
  • Prof. G. K. Garg, G. B. Pant Univ. of Agri.
    Tech., Pant Nagar, India.
Write a Comment
User Comments (0)
About PowerShow.com