Conformational Analysis of Processivity Clamps in Solution Demonstrates that Tertiary Structure Does Not Correlate with Protein Dynamics

Abstract

Summary The relationship between protein sequence, structure, and dynamics has been elusive. We report one of the first comprehensive analyses using an in-solution experimental approach to study how the conservation of tertiary structure correlates with protein dynamics. Hydrogen exchange measurements of eight processivity clamp proteins from different species revealed that, despite highly similar three-dimensional structures, clamp proteins display a wide range of dynamic behavior. Differences were apparent both for structurally similar domains within proteins and for corresponding domains of different proteins. Several of the clamps contained regions that underwent local unfolding with different half-lives. We also observed a conserved pattern of alternating dynamics of the α-helices lining the inner pore of the clamps as well as a correlation between dynamics and the number of salt bridges in these α-helices. Our observations reveal that tertiary structure and dynamics are not directly correlated and that primary structure plays an important role in dynamics.

Noncognate DNA damage prevents the formation of the active conformation of the Y‐family DNA polymerases DinB and DNA polymerase κ

“…Samples containing 3–10 μ m protein in the presence or absence of primer/template DNA (two‐fold molar excess) and dNTP (1 m m ) in 15 μL of assay buffer (25 m m Hepes, pH 7.5, 20 m m NaCl, 5 m m MgSO 4 , and 3 m m DTT) were incubated for 30 min at room temperature before addition of 0.5 μL of Sypro Orange (Invitrogen, Grand Island, NY, USA) to a final concentration of 10X. Samples were analyzed with a CFX 96 Real‐Time system (Bio‐Rad, Hercules, CA, USA), as previously described .…”

Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants

“…Technology derived from Brian Chait’s first studies has made complex analyses faster, more reliable and robust, and has enabled comparative analysis of related proteins. Our laboratory has been interested in using HX MS to compare proteins that are related in tertiary structure, but not necessarily in primary structure (e.g., [2,3]). In undertaking these types of homologous protein comparison studies, we have come to realize just how complex this exercise can be.…”

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

“…The development of dedicated automated robots and software programs for data acquisition and analysis have strongly contributed to the widespread use of HDX over the past 5 years. The increasing automation of these experiments and data analysis have not only increased their robustness and reproducibility but also allowed more ambitious analyses of very large complexes [34] and/or screening large numbers of conditions [41][42][43][44][45]. Because of these developments, HDX has become competitive with more conventional epitope-mapping methods such as phage display, explaining the increasing number of HDX studies found in the literature (reviewed in [46]), and reflecting the keen interest in HDX particularly on the part of biopharmaceutical companies [47].…”

“…Because of these developments, HDX has become competitive with more conventional epitope-mapping methods such as phage display, explaining the increasing number of HDX studies found in the literature (reviewed in [46]), and reflecting the keen interest in HDX particularly on the part of biopharmaceutical companies [47]. However, one has to keep in mind that, even if the data analysis (extraction of deuteration kinetics on each peptide) is now automated, the interpretation of the data is not always straightforward, even when high resolution structures are available [43]. In fact, when looking at differences of deuteration between two conformational states, there is no common agreement on what percentage difference is significant.…”