An Experimental Investigation of Conformational Fluctuations in Proteins G and L

Abstract

The B1 domains of streptococcal proteins G and L are structurally similar, but they have different sequences and they fold differently. We have measured their NMR spectra at variable temperature using a range of concentrations of denaturant. Many residues have curved amide proton temperature dependence, indicating that they significantly populate alternative, locally unfolded conformations. The results, therefore, provide a view of the locations of low-lying, locally unfolded conformations. They indicate approximately 4-6 local minima for each protein, all within ca. 2.5 kcal/mol of the native state, implying a locally rough energy landscape. Comparison with folding data for these proteins shows that folding involves most molecules traversing a similar path, once a transition state containing a beta hairpin has been formed, thereby defining a well-populated pathway down the folding funnel. The hairpin that directs the folding pathway differs for the two proteins and remains the most stable part of the folded protein.

NMR Characterization of the Energy Landscape of SUMO-1 in the Native-state Ensemble

“…Several studies performed by Williamson and co-workers 10-12 have shown that the alternative states thus identified represent locally unfolded forms, typically consisting of approximately five amino acid residues centered on the residue that displays curved temperature dependence. [10][11][12] As shown in Figure 5(a), in the native condition of SUMO-1, about eight residues show curvature in temperature-dependent chemical shifts. Upon addition of urea more residues seem to be participating in conformational exchange and thus acquire curved profiles.…”

NMR Investigations on Ruggedness of Native State Energy Landscape in Folded Proteins

“…The locations of the alternative states the global unfolding pathway of cytochrome c, hinted that they may determine the pathway. Similar experiments on B1 domains of streptococcal proteins G and L (Tunnicliffe et al 2005), which are structurally similar, but have different sequences and folding established that several of the residues have curved amide proton temperature and indicated approximately 4-6 local minima for each protein. Further, reports on N-terminal domain of phosphoglycerate kinase, hen eggwhite lysozyme, SUMO1 and BPTI (Baxter et al 1998;Kumar et al 2007) established that conformational heterogeneity arises from a number of independent sources such as, aromatic ring current effects, a minor conformer generated through disulphide bond isomerisation; an alternative hydrogen bond network associated with buried water molecules; alternative hydrogen bonds involving backbone amides and surface-exposed side-chain hydrogen bond acceptors; and the disruption of loops, ends of secondary structural elements and chain termini.…”

“…Extensive research investigations on theoretical and experimental aspects of different protein systems regarding low-energy excited states have been performed by Williamson and co-workers (Baxter et al 1998;Tunnicliffe et al 2005;Williamson 2003). Studies on conformational ensemble of cytochrome c revealed high structural entropy (Williamson 2003).…”

“…such as paramagnetic relaxation enhancement (PRE) (Clore and Iwahara 2009;Clore 2011), relaxation dispersion (RD) (Boehr et al 2006;Mittermaier and Kay 2006;Tzeng and Kalodimos 2009) and non-linear temperature dependence of amide proton chemical shifts (Krishna Mohan et al 2008;Mohan et al 2008b;Tunnicliffe et al 2005;Williamson 2003) have emerged and significantly contributed to our understanding of the relationship between structure, dynamics and function of proteins with respect to the excited-state conformers that are sparsely populated and often exist transiently.…”

Structure-function-folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

“…In fact, environment-sensitive residues are the ones that play prominent roles in various biological activities such as signal transduction, enzymatic catalysis, macromolecular association, cargo traffi cking, etc. Non-linear temperature dependence or curved temperature dependence of the amide proton chemical shifts provides information about the residues that access alternative states (Baxter et al 1998;Williamson 2003;Tunnicliffe et al 2005;Krishna Mohan et al 2008;Mohan et al 2008). Temperature-dependent studies on the DLC8 dimer in the range 290-315 K by giving a small pH perturbation (pH 7 to pH 6) have provided signifi cant insights into nativestate fl uctuations (Krishna Mohan et al 2008).…”