Characterizing Dynamic Protein-Protein Interactions Using Differentially Scaled Paramagnetic Relaxation Enhancement

Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 11/2009; 131(47):17291-7. DOI: 10.1021/ja906673c
Source: PubMed


Paramagnetic relaxation enhancement (PRE) is a powerful NMR technique that allows direct visualization of minor species. The PRE is obtained by conjugating a paramagnetic probe, such as EDTA-Mn(2+), at a specific cysteine residue. For a fast exchange between major and minor species, the observed PRE rate approaches population-weighted average of PRE values for both states. We have employed a tripeptide of Cu(2+)-binding paramagnetic probe that yields a much weaker PRE effect than EDTA-Mn(2+) does. We show that by using two probes of different paramagnetic strengths attached at the same site, the relative population and exchange time scale can be extracted, providing that the dynamic event occurs in the second to millisecond regime. Hence, this improved PRE scheme, differentially scaled paramagnetic relaxation enhancement (DiSPRE), permits both temporal and spatial characterization of a dynamic system. When applying the DiSPRE scheme to reassess the weak interactions between the N-terminal domain of enzyme I and phosphocarrier protein (HPr) from the bacterial phosphotransferase system, we have identified a minor species of excited-state complex with a approximately 4% population and exchanging with the stereospecific complex at approximately 1100 s(-1). Such species is distinct from other encounter complexes previously characterized and is likely a result of promiscuity of the HPr binding interface.


Available from: Alexander N Volkov
  • Source
    • "Here, in order to broaden the array of experimental techniques available for investigation of subunit exchange kinetics in oligomeric proteins, we have developed novel pulsed electron paramagnetic resonance spectroscopy (EPR) double electron–electron resonance (DEER) and nuclear magnetic resonance spectroscopy (NMR) paramagnetic relaxation enhancement (PRE) based methods for measurement of rate constants for subunit exchange. While DEER and PREs have been widely applied to protein structure determination, protein–protein and protein-DNA complex structure determination (Battiste and Wagner 2000; Borbat et al. 2002; Hilger et al. 2007; Rumpel et al. 2008; Ward et al. 2009; Yang et al. 2010, 2011), transient intermediate detection (Iwahara and Clore 2006; Tang et al. 2006), characterization of dynamics of transient macromolecular interactions (Clore et al. 2007; Tang et al. 2007; Tang et al. 2008a, b) and weak protein–protein interactions (Jeschke et al. 2006a; Yu et al. 2009), these methods have not previously been used for measurement of homodimer protein subunit exchange rate constants. We demonstrated these new experimental approaches using the homodimer protein Dsy0195 from the strictly anaerobic bacterium Desulfitobacterium hafniense Y51. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Here, we report novel methods to measure rate constants for homodimer subunit exchange using double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy measurements and nuclear magnetic resonance spectroscopy based paramagnetic relaxation enhancement (PRE) measurements. The techniques were demonstrated using the homodimeric protein Dsy0195 from the strictly anaerobic bacterium Desulfitobacterium hafniense Y51. At specific times following mixing site-specific MTSL-labeled Dsy0195 with uniformly (15)N-labeled Dsy0195, the extent of exchange was determined either by monitoring the decrease of MTSL-labeled homodimer from the decay of the DEER modulation depth or by quantifying the increase of MTSL-labeled/(15)N-labeled heterodimer using PREs. Repeated measurements at several time points following mixing enabled determination of the homodimer subunit dissociation rate constant, k (-1), which was 0.037 ± 0.005 min(-1) derived from DEER experiments with a corresponding half-life time of 18.7 min. These numbers agreed with independent measurements obtained from PRE experiments. These methods can be broadly applied to protein-protein and protein-DNA complex studies.
    Journal of Biomolecular NMR 11/2012; 55(1). DOI:10.1007/s10858-012-9685-7 · 3.14 Impact Factor
  • Source
    • "The Cc-CcP electron transfer complex is so plastic that a single conservative point mutation at the interface can yield extreme differences in orientation (Kang and Crane, 2005). Finally, recent evidence suggests that heterogeneous binding modes of a complex may even exist without mutation, as in the case of Ein-HPr, which samples a 180 flipped orientation that has only a 2 kcal/mol difference to the solved crystal structure (Yu et al., 2009). These examples highlight the fact that even in natural complexes, specificity can be extremely fine grained, with alternate orientations lying very near canonical crystallographically observed states in both evolutionary and energetic space. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.
    Molecular cell 03/2011; 42(2):250-60. DOI:10.1016/j.molcel.2011.03.010 · 14.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: There is a recent interest in X-band weather radar due to the high sensitivity and to the cheap microwave hardware. However, the attenuation due to the precipitation itself makes it difficult to estimate the rainrate unless this phenomena is of limited extent, as for insulated phenomena and/or short range operation (e.g. for valleys, urban basin hydrology). The authors analyze the limits in the rainfall rate estimation in the X-band using differential phase shift (mainly due to signal-to-noise ratio) and the effect of the backscattering differential phase shift
    Geoscience and Remote Sensing Symposium Proceedings, 1998. IGARSS '98. 1998 IEEE International; 08/1998
Show more