Efforts toward Developing Direct Probes of Protein Dynamics

Cremeens, Matthew E.; Fujisaki, Hiroya; Zhang, Yong; Zimmermann, Jörg; Sagle, Laura; Matsuda, Shigeo; Dawson, Philip E.; Straub, John E.; Romesberg, Floyd E.

doi:10.1021/ja061328g

Cited by 51 publications

(56 citation statements)

References 11 publications

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“…FT IR spectra were recorded on a Bruker Equinox 55 spectrometer equipped with a liquid nitrogen cooled MCT detector, as described previously. 36,37 The sample chamber was purged with nitrogen (14 psi) for 30 min after opening chamber and during data acquisition. Absorption spectra were obtained from measured interferograms using Bruker OPUS software.…”

Section: Methodsmentioning

confidence: 99%

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

2008

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Much effort has been directed towards understanding the contributions of electrostatics and dynamics to protein function and especially to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct experimental probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biological catalysis. We observe that the stretching absorption frequency of (methyl-d 3 ) methionine carbon-deuterium bonds show an approximately linear dependence on solvent dielectric. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary analysis of (methyl-d 3 ) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics, and potentially, their contribution to catalysis.

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Section: Methodsmentioning

confidence: 99%

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

2008

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“…By combining Equations 5 and 6, the linear relationship between enzyme and experimental rate constant: (8) can be modeled for reactions with low concentration of enzyme. This is termed "cleavagelimited conditions", and data from such reactions can be fit with a linear regression ( Figure 3B).…”

Section: Kinetic Models Of Proteolysismentioning

confidence: 99%

“…A range of biophysical approaches can be used to describe the energy landscape of a protein (i.e. the distributions of different conformations and the rates of exchange), and this has been done for a number of monomeric proteins (8)(9)(10). However, due to technical challenges, detailed mapping is not available for the dynamics of viruses, or any other, megadalton complexes.…”

Section: Introductionmentioning

confidence: 99%

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

Hilmer

Zlotnick

Bothner

2008

Journal of Molecular Biology

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Functional analysis of Hepatitis B virus (HBV) core particles has associated a number of biological roles with the C-terminus of the capsid protein. One set of functions require the C-terminus to be on the exterior of the capsid, while others place this domain on the interior. According to the crystal structure of the capsid, this segment is strictly internal to the capsid shell and buried at a proteinprotein interface. Using kinetic hydrolysis, a form of protease digestion assayed by SDS-PAGE and mass spectrometry, the structurally and biologically important C-terminal region of HBV capsid protein assembly domain (Cp149, residues 1-149) has been shown to be dynamic in both dimer and capsid forms. HBV is an enveloped virus with a T=4 icosahedral core that is composed of 120 copies of a homodimer capsid protein. Free dimer and assembled capsid forms of the protein are readily hydrolyzed by trypsin and thermolysin, around residues 127-128, indicating that this region is dynamic and exposed to the capsid surface. The measured conformational equilibria have an opposite temperature dependence between free dimer and assembled capsid. This work helps to explain the previously described allosteric regulation of assembly and functional properties of a buried domain. These observations make a critical connection between structure, dynamics, and function: made possible by the first quantitative measurements of conformational equilibria and rates of conversion between protein conformers for a megadalton complex.

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“…[21][22][23][24][25][26][27][28] These methods have been applied to answer basic questions in protein folding and function, which were otherwise difficult to answer with expressed proteins containing native sequences and naturally occurring amino acids. [29][30][31] Earlier versions of NCL were limited by the requirement for cysteine at the ligation junction, as well as a strong dependence on peptide sequence at the ligation junction, primarily due to steric influence on ligation yield. The invention of removable thiol auxiliaries [27,[32][33][34][35] has expanded the sequence repertoire that is accessible to NCL, and recently the new methods have been applied to synthesis of full-length glycoproteins [36] or fragments thereof.…”

Section: Introductionmentioning

confidence: 99%

Enzymes for Glycoprotein Synthesis

Wong¹

2009

Chimia

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More than 90% of human proteins are glycosylated and since protein glycosylation is understood to play a role in folding, trafficking, stability, immunogenicity, and function there is a need to generate pure protein glycoforms. Pure single protein glycoforms are difficult to obtain because the cellular machinary produces complex mixtures for any given protein. In this article an overview is given of various approaches used to generate specific single glycoforms of proteins, studies of the role of glycosylation in protein stability and function, and the imaging and identification of new glycoproteins related to cancer.

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Efforts toward Developing Direct Probes of Protein Dynamics

Cited by 51 publications

References 11 publications

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

Enzymes for Glycoprotein Synthesis

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