# Presentation: 035223

## 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@ncifcrf.gov**

## 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. **

## 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.**

## 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 protein’s environment, *****e. g.,***** temperature, pH, salt, solvent, presence/absence of protein’s substrates, ligands or denaturants (Urea, GdmHCl).**
**Localization in the cell.**

## 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. **

## 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 protein’s stability as a function of temperature yields protein stability curve described by Gibbs-Helmoholtz equation:**
******G(T) = ********H****G**** (1-T/T****G****) - ********C****p**** [(T****G**** - T) + T ln(T/T****G****)]**
**The parameters T****G****, ********H****G ****and ********C****p  ****are determined using experimental procedures.**

## A protein stability curve yields several cardinal characteristics of the protein

**Slope at T****G**** = -********H****G ****/ T****G**** = - ********S****G**
**Curvature at T****G**** = -********C****p ****/ T****G**
** ****Temperature of maximal protein stability, **
**      ****T****S**** = T****G**** exp (-********H****G  ****/ T****G**** ********C****p****)**
**Maximal protein stability,**
**    ********G(T****S****) = ********H****G**** - (T****G**** – T****S****) ********C****p**
**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.**** **

## 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. **

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

## 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.

## 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.**

## 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).**

## 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?

**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?**

## 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, ****q****i**** ****and ****q****j****.  **
**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. **

- .**q****i**

- .**q****j**

****** ****= 4**

******=80**

## 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.

**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.**

## Slide 16

## 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 T****G**** of 113********C while CsGDH has T****G**** 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.**

## 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. **

## 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.**

## 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. **

## 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.**

## Slide 22

## 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.**

## 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 C******** atom and a side chain charged group centroid in an ion pairing residue. We actually take the supplemental angle.**

## 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.

**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.**

## 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.**

## 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? **

**Notes:**
PfCs and DsCs 41% seq. identity. C-alpha RMSD 1.26 Ang.
DsCs and GgCs 28.7% seq. identity. C-alpha RMSD 1.68 Ang.

## 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 0°C and 100°C.**
**The electrostatic free energy contributions by individual charged residues show greater fluctuations in the psychrophilic citrate synthase.**

## Slide 29

## 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.**

## 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.**