Project 1: Structure-function-disease analysis of dystrophin and utrophin in muscular dystrophy

Muscular dystrophy (MD) refers to a group of degenerative muscle diseases that cause progressive muscle weakness. MD affects all types of muscles. For example, decreased function of heart muscles causes heart diseases that include cardiomyopathy and congestive heart failure. Duchenne MD (DMD) and Becker MD (BMD) are two prominent types of MD, which are caused by the deficiency of a vital muscle protein known as dystrophin. These dystrophin-related diseases physically weaken patients to a state of immobility, and often cause death at an early age. Dystrophin stabilizes the sarcolemma membrane against the mechanical forces associated with muscle contraction and stretch. Mutations in dystrophin trigger the disease. Although dystrophin was identified as a key molecular player in MD 30 years ago, little is known about the biophysical mechanisms that trigger the disease at the fundamental protein level.

Utrophin, the closest homologue of dystrophin (60% sequence similarity), has been shown to compensate for the loss of functional dystrophin in animal studies, but its exact biological function is not known. It binds to actin, protects actin against its depolymerization, and interacts with dystrophin-related proteins. Utrophin is confined specifically to the sarcolemma in fetal and regenerating muscle cells. After down-regulation at birth, it is only found in the neuromuscular junctions in adult muscle cells to aid in optimal synapse transmission and to play a stabilizing role at these junctions.

In this project, we are trying to understand the biophysical and structural principles of how these two important proteins function, the effect thereon of disease-causing mutations, and whether we can develop new therapies based on the fundamental understanding of structure-function of dystrophin and utrophin.

Recent Publications

  • S.M. Singh, S. Bandi, and K.M.G. Mallela, The N-terminal flanking region modulates the actin binding affinity of the utrophin tandem calponin-homology domain, Biochemistry, 56 (2017) 2627-2636. PDF
  • S.M. Singh, S. Bandi, and K.M.G. Mallela, The N- and C-terminal domains differentially contribute to the structure and function of dystrophin and utrophin tandem calponin-homology domains, Biochemistry, 54 (2015) 6942-6950. PDF
  • S. Bandi, S.M. Singh, and K.M.G. Mallela, Interdomain linker determines primarily the structural stability of dystrophin and utrophin tandem calponin-homology domains rather than their actin-binding affinity, Biochemistry, 54 (2015) 5480-5488. PDF
  • S.M. Singh, S. Bandi, D.D. Shah, G. Armstrong, and K.M.G. Mallela, Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function, PLoS One, 9 (2014) e110439. PDF
  • S. Bandi, S.M. Singh, and ;K.M.G. Mallela, The C-terminal domain of the utrophin tandem calponin-homology domain appears to be thermodynamically and kinetically more stable than the full-length protein, Biochemistry, 53 (2014) 2209-2211. PDF

Project 2: Mechanisms of excipient interactions with pharmaceutical proteins

Excipients play a major role in formulating a drug substance into a drug product. These include antimicrobial preservatives such as benzyl alcohol to prevent the accidental growth of microbes in protein formulations, aggregation suppressors such as polysorbates, reactive oxygen scavengers such as methionine, surface deadsorbents such as silicone oil, and others. In principle, excipients should be inert substances that should merely serve as the vehicle or medium for a drug or active substance, but in reality, these can interact with protein drugs causing unwanted protein destabilization and aggregation. In this project, we are trying to understand the fundamental biophysical and structural mechanisms by which excipients interact with pharmaceutical proteins using a suite of biophysical techniques that include far-UV and near-UV circular dichroism, fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and 2D NMR. This work is being done as part of our Center for Pharmaceutical Biotechnology, and please contact us for any future collaborations.

Recent Publications

  • D. Shah, J. Zhang, H. Maity, and K.M.G. Mallela, Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody, International Journal of Pharmaceutics, 547 (2018) 438-449. PDF
  • S.M. Singh, S. Bandi, D.N.M. Jones, and K.M.G. Mallela, Effect of polysorbate 20 and polysorbate 80 on the higher-order structure of a monoclonal antibody and its Fab and Fc fragments probed using 2D nuclear magnetic resonance spectroscopy, Journal of Pharmaceutical Sciences, 106 (2017) 3486-3498. PDF
  • R.L. Bis, S.M. Singh, J. Cabello-Villegas, and K.M.G. Mallela, Role of benzyl alcohol in the unfolding and aggregation of interferon alpha-2a, Journal of Pharmaceutical Sciences, 104 (2015) 407-415. PDF
  • R.L. Bis and K.M.G. Mallela, Antimicrobial preservatives induce aggregation of interferon alpha-2a: The order in which preservatives induce protein aggregation is independent of the protein, International Journal of Pharmaceutics, 472 (2014) 356-361. PDF
  • R.L. Bis, T.M. Stauffer, S.M. Singh, T.B. Lavoie, and K.M.G. Mallela, High yield soluble bacterial expression and streamlined purification of recombinant human interferon alpha-2a, Protein Expression and Purification, 99 (2014) 138-146. PDF
  1. D. Shah, J. Zhang, H. Maity, and K.M.G. Mallela, Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody, International Journal of Pharmaceutics, 547 (2018) 438-449.  Abstract • Full text • PDF   
  2. D. Shah, S.M. Singh, M. Dzieciatkowska, and K.M.G Mallela, Biophysical analysis of the effect of chemical modification by 4-oxononenal on the structure, stability, and function of binding immunoglobulin protein (BiP), PLoS One, 12 (2017) e0183975.  Abstract • Full text • PDF  
  3. S.M. Singh, S. Bandi, D.N.M. Jones, and K.M.G. Mallela, Effect of polysorbate 20 and polysorbate 80 on the higher-order structure of a monoclonal antibody and its Fab and Fc fragments probed using 2D nuclear magnetic resonance spectroscopy, Journal of Pharmaceutical Sciences, 106 (2017) 3486-3498.  Abstract • Full text • PDF  
  4. S.M. Singh, S. B​andi, and K.M.G. Mallela, The N-terminal flanking region modulates the actin binding affinity of the utrophin tandem calponin-homology domain, Biochemistry, 56 (2017) 2627-2636.  Abstract • Full text • PDF  
  5. S.M. Singh, S. Bandi, and K.M.G. Mallela, The N- and C-terminal domains differentially contribute to the structure and function of dystrophin and utrophin tandem calponin-homology domains, Biochemistry, 54 (2015) 6942-6950. Abstract  Full text  PDF
  6. S. Bandi, S.M. Singh, and K.M.G. Mallela, Interdomain linker determines primarily the structural stability of dystrophin and utrophin tandem calponin-homology domains rather than their actin-binding affinity, Biochemistry, 54 (2015) 5480-5488. Abstract  Full text  PDF  
  7. R.L. Bis, S.M. Singh, J. Cabello-Villegas, and K.M.G. Mallela, Role of benzyl alcohol in the unfolding and aggregation of interferon alpha-2a, Journal of Pharmaceutical Sciences, 104 (2015) 407-415. AbstractFull textPDF
  8. S.M. Singh, S. Bandi, D.D. Shah, G. Armstrong, and K.M.G. Mallela, Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function, PLoS One, 9 (2014) e110439. AbstractFull textPDF.
  9. R.L. Bis and K.M.G. Mallela, Antimicrobial preservatives induce aggregation of interferon alpha-2a: The order in which preservatives induce protein aggregation is independent of the protein, International Journal of Pharmaceutics, 472 (2014) 356-361. AbstractFull textPDF
  10. R.L. Bis, T.M. Stauffer, S.M. Singh, T.B. Lavoie, and K.M.G. Mallela, High yield soluble bacterial expression and streamlined purification of recombinant human interferon alpha-2a, Protein Expression and Purification, 99 (2014) 138-146. AbstractFull textPDF
  11. S. Bandi, S.M. Singh, and K.M.G. Mallela, The C-terminal domain of the utrophin tandem calponin-homology domain appears to be thermodynamically and kinetically more stable than the full-length protein, Biochemistry, 53 (2014) 2209-2211. AbstractFull TextPDF
  12. S.M. Singh, S. Bandi, S.J. Winder, and K.M.G. Mallela, The actin binding affinity of the utrophin tandem calponin-homology domain is primarily determined by its N-terminal domain, Biochemistry, 53 (2014) 1801-1809. AbstractFull TextPDF
  13. R.L. Hutchings, S.M. Singh, J. Cabello-Villegas, and K.M.G. Mallela, Effect of antimicrobial preservatives on partial protein unfolding and aggregation, Journal of Pharmaceutical Sciences, 102 (2013) 365-376. AbstractFull textPDF
  14. S.M. Singh and K.M.G. Mallela, The N-terminal actin-binding tandem calponin-homology (CH) domain of dystrophin is in a closed conformation in solution and when bound to F-actin, Biophysical Journal, 103 (2012) 1970-1978. AbstractFull textPDF. Selected for a New & Notable: Biophysical Journal, 103 (2012) 1818-1819. Full textPDF
  15. S.M. Singh, J.F. Molas, N. Kongari, S. Bandi, G.S. Armstrong, S.J. Winder, and K.M.G. Mallela, Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin binding domains of utrophin and dystrophin, Proteins: Structure, Function, and Bioinformatics, 80 (2012) 1377-1392. AbstractFull textPDF
  16. S.M. Singh, R.L. Hutchings, and K.M.G. Mallela, Mechanisms of m-cresol induced protein aggregation studied using a model protein cytochrome c, Journal of Pharmaceutical Sciences, 100 (2011) 1679-1689. AbstractFull textPDF
  17. S.M. Singh, N. Kongari, J. Cabello-Villegas, and K.M.G. Mallela, Mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-beta aggregates, Proceedings of the National Academy of Sciences of the United States of America, 107 (2010) 15069-15074. AbstractFull textPDF
  18. S.M. Singh, J. Cabello-Villegas, R.L. Hutchings, and K.M.G. Mallela, Role of partial protein unfolding in alcohol-induced protein aggregation, Proteins: Structure, Function, and Bioinformatics, 78 (2010) 2625-2637. AbstractFull textPDF
  19. K.M.G. Mallela, Pharmaceutical Biotechnology – Concepts and Applications, Human Genomics, 4 (2010) 218-219. Full textPDF
  20. S. Bédard, M.M.G. Krishna, L. Mayne, and S.W. Englander, Protein folding: Independent unrelated pathways or predetermined pathway with optional errors, Proceedings of the National Academy of Sciences of the United States of America, 105 (2008) 7182-7187. (Equal contribution) AbstractFull textPDF
  21. S.W. Englander, L. Mayne, and M.M.G. Krishna, Protein folding and misfolding: Mechanism and principles, Quarterly Reviews of Biophysics, 40 (2007) 287-326. AbstractFull textPDF
  22. M.M.G. Krishna, H.Maity, J.N. Rumbley, and S.W. Englander, Branching in the sequential folding pathway of cytochrome c, Protein Science, 16 (2007) 1946-1956. AbstractFull textPDF
  23. M.M.G. Krishna and S.W. Englander, A unified mechanism for protein folding: Predetermined pathways with optional errors, Protein Science, 16 (2007) 449-464. AbstractFull textPDF 
  24. M.M.G. Krishna, H. Maity, J.N.Rumbley, Y. Lin, and S.W. Englander, Order of steps in the cytochrome c folding pathway: Evidence for a sequential stabilization mechanism, Journal of Molecular Biology, 359 (2006) 1410-1419. AbstractJournal coverFull textPDF
  25. R. Pidikiti, T. Zhang, K.M.G. Mallela, M. Shamim, K.S. Reddy, and J.S. Johansson, Sevoflurane-induced structural changes in a four-alpha-helix bundle protein, Biochemistry, 44 (2005) 12128-12135. AbstractFull textPDF
  26. R. Pidikiti, T. Zhang, K.M.G. Mallela, M. Shamim, K.S. Reddy, and J.S. Johansson, Structural changes in a four-alpha-helix bundle protein following sevoflurane binding, International Congress Series, 1283 (2005) 155-159. AbstractFull textPDF
  27. R. Pidikiti, M. Shamim, K.M.G. Mallela, K.S. Reddy, and J.S. Johansson, Expression and characterization of a four-alpha-helix bundle protein that binds the volatile general anesthetic halothane, Biomacromolecules, 6 (2005) 1516-1523. AbstractFull textPDF
  28. H. Maity, M. Maity, M.M.G. Krishna , L. Mayne, and S.W. Englander, Protein folding: The stepwise assembly of foldon units, Proceedings of the National Academy of Sciences of the United States of America, 102 (2005) 4741-4746. AbstractFull textPDF
  29. M.M.G. Krishna and S.W. Englander, The N-terminal to C-terminal motif in protein folding and function, Proceedings of the National Academy of Sciences of the United States of America, 102 (2005) 1053-1058. AbstractFull textPDF
  30. M.M.G. Krishna, Y. Lin, and S.W. Englander, Protein misfolding: Optional barriers, misfolded intermediates, and pathway heterogeneity, Journal of Molecular Biology, 343 (2004) 1095-1109. AbstractFull textPDF
  31. M.M.G. Krishna, L. Hoang, Y. Lin, and S.W. Englander, Hydrogen exchange methods to study protein folding, Methods, 34 (2004) 51-64. AbstractFull textPDF
  32. M.M.G. Krishna, Y. Lin, L. Mayne, and S.W. Englander, Intimate view of a kinetic protein folding intermediate: Residue-resolved structure, interactions, stability, folding and unfolding rates, homogeneity, Journal of Molecular Biology, 334 (2003) 501-513. AbstractFull textPDF
  33. L. Hoang, H. Maity, M.M.G. Krishna, Y. Lin, and S.W. Englander, Folding units govern the cytochrome c alkaline transition, Journal of Molecular Biology, 331 (2003) 37-43. AbstractFull textPDF
  34. M.M.G. Krishna, Y. Lin, J. Rumbley, and S.W. Englander, Cooperative omega loops in cytochrome c: Role in folding and function, Journal of Molecular Biology, 331 (2003) 29-36. AbstractFull textPDF
  35. L. Hoang, S. Bédard, M.M.G. Krishna, Y. Lin, and S.W. Englander, Cytochrome c folding pathway: Kinetic native-state hydrogen exchange, Proceedings of the National Academy of Sciences of the United States of America, 99 (2002) 12173-12178. AbstractFull textPDF
    Corrections: Proceedings of the National Academy of Sciences of the United States of America, 99 ( 2002) 15831. Full textPDF
  36. M.M.G. Krishna, Studying how a protein folds, The Alchemist (Web journal of chemweb.com), 2002. PDF
  37. S.W. Englander and M.M.G. Krishna, Hydrogen exchange, Nature Structural Biology, 8 (2001) 741-742. Full textPDF
  38. A.S.R. Koti, M.M.G. Krishna, and N. Periasamy, Time-resolved area-normalized emission spectroscopy (TRANES): A novel method for confirming emission from two excited states, Journal of Physical Chemistry A 105 (2001) 1767-1771. AbstractFull textPDF
  39. M.M.G. Krishna, A. Srivastava, and N. Periasamy, Rotational dynamics of surface probes in lipid vesicles, Biophysical Chemistry, 90 ( 2001) 123-133. AbstractFull textPDF
    Erratum: Biophysical Chemistry, 91 (2001) 209. Full textPDF
  40. A. Mishra, G.B. Behera, M.M.G. Krishna, and N. Periasamy, Time-resolved fluorescence studies of aminostyrylpyridinium dyes in organic solvents and surfactant micelles, Journal of Luminescence, 92 (2001) 175-188. AbstractFull textPDF
  41. M.M.G. Krishna, J. Samuel, and S. Sinha, Brownian motion on a sphere: Distribution of solid angles, Journal of Physics A: Mathematical and General, 33 (2000) 5965-5971. AbstractFull textPDF
    Also on ArXiv: http://arxiv.org/abs/cond-mat/0005345v3 PDF
  42. M.M.G. Krishna, R. Das, N. Periasamy, and R. Nityananda, Translational diffusion of fluorescent probes on a sphere: Monte Carlo simulations, theory and fluorescence anisotropy experiment, Journal of Chemical Physics, 112 (2000) 8502-8514. AbstractFull textPDF
  43. M.M.G. Krishna , Dynamics of Fluorescent Probes in Biological Systems, PhD Thesis, Tata Institute of Fundamental Research, University of Mumbai, India, 1999. PDF  © M.M.G. Krishna & N. Periasamy. Please cite the thesis when referring to unpublished results.
  44. M.M.G. Krishna and N. Periasamy, Location and orientation of DODCI in lipid bilayer membranes: Effects of lipid chain length and unsaturation, Biochimica et Biophysica Acta (Biomembranes), 1461 (1999) 58-68. AbstractFull textPDF
  45. M.M.G. Krishna, Excited state kinetics of the hydrophobic probe nile red in membranes and micelles, Journal of Physical Chemistry A, 103 (1999) 3589-3595. AbstractFull textPDF
    Additions and Corrections: Journal of Physical Chemistry A, 103 (1999) 4129. Full textPDF
    More Corrections: PDF
  46. M.M.G. Krishna and N. Periasamy, Orientational distribution of linear dye molecules in bilayer membranes, Chemical Physics Letters, 298 (1998) 359-367. AbstractFull textPDF
  47. M.M.G. Krishna, V.K. Rastogi, N. Periasamy, and K.V.R. Chary, Fluorescence and NMR studies on human seminal plasma prostatic inhibin: Association of lifetimes with sterically constrained tryptophans, Journal of Physical Chemistry B, 102 (1998) 5520-5528. AbstractFull textPDF
  48. M.M.G. Krishna and N. Periasamy, Fluorescence of organic dyes incorporated in lipid membranes: Site of solubilization and the effects of viscosity and refractive index on lifetimes, Journal of Fluorescence, 8 (1998) 81-91. AbstractFull textPDF
  49. N.C. Maiti, M.M.G. Krishna, P.J. Britto, and N. Periasamy, Fluorescence dynamics of dye probes in micelles, Journal of Physical Chemistry B, 101 (1997) 11051-11060. AbstractFull textPDF
  50. M.M.G. Krishna and N. Periasamy, Spectrally constrained global analysis of fluorescence decays in biomembrane systems, Analytical Biochemistry, 253 (1997) 1-7. AbstractFull textPDF

For comments, suggestions and complaints, please email Krishna.Mallela@CUAnschutz.edu.

The PDF files provided on this page are strictly for scientific, non-commercial research only. For all other purposes, the users should contact the respective copyright holders.​

  

Dinen Shah: Graduate Student
Phone: 303-724-3577
Email: Dinen.Shah@ucdenver.edu

Dinen’s current research is on determining the effect​s of lipid peroxidation products on the structure and function of a heat shock protein, 78 kDa glucose-regulated protein (GRP78), and the effect of chemical modifications such as oxidation and monoclonal antibodies.

Shah_Dinen

Scott Pardee: Graduate Student
Phone: 303-724-3577
Email:Scott.Pardee@CUAnschutz.edu

Scott's current research is on excipient interactions with pharmaceutical proteins.

ScottPardee

Dr. Swati Bandi: Postdoctoral Fellow
Phone: 303-724-3577
Email: Swati.Bandi@CUAnschutz.edu

Swati’s current research is on characterizing the structural dynamics of tandem calponin-homology domains of dystrophin and utrophin, and polysorbate interactions with monoclonal antibodies.

Bandi_Swati

Dr. Vaibhav Upadhyay: Postdoctoral Fellow
Phone: 303-724-3577
Email: Vaibhav.Upadhyay@CUAnschutz.edu

Vaibhav’s current research is on characterizing the structure-function of dystrophin and utrophin.

VaibhavUpadhyay
  

​​Dr. Regina L. Bis: Postdoctoral Fellow
Phone: 303-724-3577
Email: Regina.Bis@ucdenver.edu

Regina’s current research is on determining the mechanisms of alcohol and antimicrobial-preservative-induced aggregation of a model protein cytochrome c and a pharmaceutical protein interferon alpha-2a.

Bis_Regina

Justine F. Molas: Graduate Student

Justine optimized the expression and purification protocols of dystrophin and utrophin, and did initial biophysical characterization of these proteins.

Darin Brown: Graduate Student

Darin worked on the expression and purification of death domains, Fas, FADD, DR4 & DR5, which are involved in apoptotic signaling pathways.

Joseph Rower: Graduate Student

Joseph worked on the effect of Hofmeister series on thermal aggregation of cytochrome c.

Dr. Surinder M. Singh: Postdoctoral Fellow
Phone: 303-724-3577
Email: Surinder.Singh@ucdenver.edu

Surinder’s current research is on characterizing the biophysics of dystrophin and utrophin, two key proteins involved in muscular dystrophy.

Geoffrey Armstrong, University of Colorado Boulder
NMR experiments on utrophin and dystrophin

David Fela, NPS Pharmaceuticals
Biophysical characterization of parathyroid hormone (PTH) using NMR

Vivian Hook, University of California San Diego
Dystrophin expression in muscle cells

Thomas Lavoie, PBL Assay Science
Functional assays on interferon alpha-2a

David Nesbitt, University of Colorado Boulder
Fluorescence experiments on dystrophin and utrophin

Tara Stauffer, PBL Assay Science
Antiviral and antiproliferative assays on interferon alpha-2a

Steve Winder, University of Sheffield
Expression vectors of dystrophin and utrophin N-ABDs, and their purification and actin-binding assays

Deborah Wuttke, University of Colorado Boulder
NMR experiments on dystrophin and utrophin

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