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.