Casey H. Londergan

Haverford College, Norristown, PA, United States

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Publications (25)86.07 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Symmetric and asymmetric crystal structures of the apo and transition state analog forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active site cysteine-283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analog forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in both the presence and the absence of the substrate. The S-H peak persists at all physiologically relevant pH, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule, and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multi-population CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared lineshape. Together, these spectral observations localized at the active site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.
    Biochemistry 10/2014; · 3.38 Impact Factor
  • Kevin W Hoffman, Matthew G Romei, Casey H Londergan
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    ABSTRACT: The amino acid histidine (His) has a number of unique roles that can dictate function in proteins, and these roles are typically conferred through noncovalent interactions that depend on the protonation state of His's 4-substituted imidazole ring. His's protonation state can vary near physiological pH, and a probe of His's variable protonation state and its resulting noncovalent interactions that has both high time resolution and no sample limitations could find wide use in determining the role of particular His residues in proteins. Here we use a classic deuterium exchange reaction to replace the C2-H hydrogen atom of the His imidazole ring with deuterium, leading to a unique aromatic C2-D stretching vibration whose frequency is sensitive to environmental changes across the entire imidazole ring. Using non-resonant Raman spectroscopy, we demonstrate using model compounds that the frequency of this C2-D vibration shifts by 35 cm-1 upon changes in the His protonation state. The C2-D band is a very weak infrared absorber, so this vibration is not expected to be useful in infrared transmission experiments for proteins. Solvent-dependent Raman experiments indicate that the C2-D band of the neutral imidazole ring is sensitive to H-bonding interaction with donors and acceptors of varying strengths, suggesting that the C2-D frequency can be used to identify H-bonding partners of specific His residues. Raman spectra at varying concentrations of Cu2+ also show the C2-D band's sensitivity to metal coordination, with differences due changes in the coordination environment. The strong Raman signal of this band and the sampling flexibility of Raman spectroscopy suggest that this vibration could be very useful in documenting the local role of His residues in many His-containing proteins and protein assemblies.
    The Journal of Physical Chemistry A 03/2013; · 2.77 Impact Factor
  • Matthew G. Romei, Kevin W. Hoffman, Casey H. Londergan
    Biophysical Journal 01/2013; 104(2):685-. · 3.67 Impact Factor
  • Biophysical Journal 01/2013; 104(2):93-. · 3.67 Impact Factor
  • Casey H. Londergan, Alice R. Vienneau, Shannon R. Dalton
    Biophysical Journal 01/2013; 104(2):54-. · 3.67 Impact Factor
  • Biophysical Journal 01/2013; 104(2):685-. · 3.67 Impact Factor
  • Rachel Baskin, Casey Londergan
    Biophysical Journal 01/2013; 104(2):332-. · 3.67 Impact Factor
  • Marta P. Wolfshorndl, Casey H. Londergan
    Biophysical Journal 01/2013; 104(2):564-. · 3.67 Impact Factor
  • Biophysical Journal 01/2013; 104(2):225-. · 3.67 Impact Factor
  • Biophysical Journal 01/2013; 104(2):234-. · 3.67 Impact Factor
  • Hailiu Yang, Johnny Habchi, Sonia Longhi, Casey H Londergan
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    ABSTRACT: The fast intrinsic time scale of infrared absorption and the sensitivity of molecular vibrational frequencies to their environments can be applied with site-specificity by introducing the artificial amino acid β-thiocyanatoalanine, or cyanylated cysteine, into chosen sites within intrinsically disordered proteins. This amino acid can be inserted through native chemical ligation at single cysteines introduced via site-directed mutagenesis. The CN stretching band of cyanylated cysteine is sensitive to local changes in both structural content and solvent exposure. This dual sensitivity makes cyanylated cysteine an especially useful probe of binding-induced structural transitions in IDPs. The general strategy of creating single-site cysteine mutations and chemically modifying them to create the vibrational chromophore, as well as observation, processing and analysis of the CN stretching band, is presented.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 895:245-70. · 1.29 Impact Factor
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    ABSTRACT: Covalently bound azido groups are found in many commercially available biomolecular precursors and substrates, and the NNN asymmetric stretching band of these groups is a strong infrared absorber that appears in a spectral region clear of other signals. In order to evaluate comprehensively the solvatochromism of the asymmetric azido NNN stretching band for site-specific use in biomolecular contexts, infrared spectra of the model compounds 5-azido,1-pentanoic acid and 3-(p-azidophenyl),1-propanoic acid were acquired in a large variety of nonpolar, polar, and hydrogen-bond-donating solvents, as well as mixed aqueous-organic solvents. Spectra in pure solvents indicated that the aliphatic NNN stretching frequency maximum does not depend on solvent polarity, while the aromatic NNN frequency displays a weak but nonzero sensitivity to polarity. In both cases, the NNN frequency exhibits a blue-shift in H-bond-donating solvents, but the frequency in water is higher than in any other H-bond-donating solvent including solvents that are stronger H-bond donors. In nonfluorinated H-bond donor solvents, the frequency blue shift scales with the density of H-bond donors. This sensitivity to the presence of water was further explored in several mixed solvent environments, with the conclusion that this vibrational mode is a highly specific sensor of hydration, even in environments containing other H-bond donors like amides and alcohols, due to the very high local density of H-bond donors in water. The relatively uncomplicated (compared to nitriles, for example), water-specific response of this vibrational mode should lead to its adoption as a site-specific probe of hydration in many different possible systems in which the presence and role of molecular water is of primary interest.
    The Journal of Physical Chemistry B 12/2011; 116(3):1172-9. · 3.61 Impact Factor
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    ABSTRACT: The synthetic antimicrobial peptide CM15, a hybrid of N-terminal sequences from cecropin and melittin peptides, has been shown to be extremely potent. Its mechanism of action has been thought to involve pore formation based on prior site-directed spin labeling studies. This study examines four single-site β-thiocyanatoalanine variants of CM15 in which the artificial amino acid side chain acts as a vibrational reporter of its local environment through the frequency and line shape of the unique CN stretching band in the infrared spectrum. Circular dichroism experiments indicate that the placements of the artificial side chain have only small perturbative effects on the membrane-bound secondary structure of the CM15 peptide. All variant peptides were placed in buffer solution, in contact with dodecylphosphatidylcholine micelles, and in contact with vesicles formed from Escherichia coli polar lipid extract. At each site, the CN stretching band reports a different behavior. Time-dependent attenuated total reflectance infrared spectra were also collected for each variant as it was allowed to remodel the E. coli lipid vesicles. The results of these experiments agree with the previously proposed formation of toroidal pores, in which each peptide finds itself in an increasingly homogeneous and curved local environment without apparent peptide-peptide interactions. This work also demonstrates the excellent sensitivity of the SCN stretching vibration to small changes in the peptide-lipid interfacial structure.
    Biochemistry 11/2011; 50(51):11097-108. · 3.38 Impact Factor
  • Biophysical Journal 02/2011; 100(3). · 3.67 Impact Factor
  • Biophysical Journal 01/2011; 100. · 3.67 Impact Factor
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
  • Biophysical Journal 01/2011; 100. · 3.67 Impact Factor
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    ABSTRACT: Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) were cyanylated at cysteine and their infrared spectra in the C triple bond N stretching region were recorded both in the absence and in the presence of one of the physiological partners of N(TAIL), namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within N(TAIL), the C triple bond N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level.
    Biophysical Journal 09/2010; 99(5):1676-83. · 3.67 Impact Factor
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    ABSTRACT: Cyanylated cysteine, or beta-thiocyanatoalanine, is an artificial amino acid that can be introduced into peptides and proteins by post-translational chemical modification of solvent-exposed cysteine side chains, and thus it can be used in any protein with a suitable expression and mutagenesis system. In this study, cyanylated cysteine is introduced at selected sites in two model peptides that have been shown to bind to membrane interfaces: a membrane-binding sequence of the human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular dichroism indicates that the secondary structures of the bound peptides are not influenced by introduction of the artificial side chain. Infrared spectra of both systems in buffer and exposed to dodecylphosphocholine micelles indicate that the CN stretching absorption band of cyanylated cysteine can clearly distinguish between membrane burial and solvent exposure of the artificial side chain. Since infrared spectroscopy can be applied in a wide variety of lipid systems, and since cyanylated cysteine can be introduced into proteins of arbitrary size via mutagenesis and post-translational modification, this new probe could see wide use in characterizing the protein-lipid interactions of membrane proteins.
    Journal of Physical Chemistry Letters 03/2010; 1(5):850-855. · 6.59 Impact Factor
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    ABSTRACT: Beta-thiocyanatoalanine, or cyanylated cysteine, is an artificial amino acid that can be introduced at solvent-exposed cysteine residues in proteins via chemical modification. Its facile post-translational synthesis means that it may find broad use in large protein systems as a probe of site-specific structure and dynamics. The C[triple bond]N stretching vibration of this artificial side chain provides an isolated infrared chromophore. To test both the perturbative effect of this side chain on local secondary structure and its sensitivity to structural changes, three variants of a model water-soluble alanine-repeat helix were synthesized containing cyanylated cysteine at different sites. The cyanylated cysteine side chain is shown to destabilize, but not completely disrupt, the helical structure of the folded peptide when substituted for alanine. In addition, the [triple bond]N stretching bandwidth of the artificial side chain is sensitive to the helix-coil structural transition. These model system results indicate that cyanylated cysteine can be placed into protein sequences with a native helical propensity without destroying the helix, and further that the CN probe may be able to report local helix formation events even when it is water-exposed in both the ordered and disordered conformational states. These results indicate that cyanylated cysteine could be a widely useful probe of structure-forming events in proteins with large in vitro structural distributions.
    The Journal of Physical Chemistry B 03/2010; 114(14):4931-6. · 3.61 Impact Factor