Structureanddynamicsofprotein excited stateswithmillisecond lifetimes

| An in-depth understanding of biological processes often requires detailed atomic resolution structures of the molecules involved. However in solution where most of these processes occur the conformation of biomolecules like RNA, DNA and proteins is not static but fluctuates. Routinely used structural techniques like X-ray crystallography, NMR spectroscopy and cryo-electron microscopy have almost always been used to determine the structure of the dominant conformation or obtain an average structure of the biomolecule in solution with very little detailed information regarding the dynamics of these molecules in solution. Over the last few years, NMR based methods have been developed to study the dynamics of these biomolecules in solution in a site-specific manner with the aim of generating structures of the different conformations that these molecules can adopt in solution. One powerful technique is the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment, which can be used to detect and characterize protein excited states that are populated for as less as 0.5% of the time with ∼0.5–10 millisecond lifetimes. Due to recent advances in NMR pulse sequences and labeling methodology, it is now possible to determine the structures of these transiently populated excited states with millisecond lifetimes by obtaining accurate chemical shifts, residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) of these excited states. In these excited states the dynamics of some methyl containing residues can also be studied. Introduction Biological macromolecules like proteins and nucleic acids are not rigid but populate a range of conformations [1–3]. These conformational fluctuations occur on a wide range of timescales ranging from picoseconds (ps) to several seconds. Several processes like ligand binding, enzymatic catalysis, protein folding etc occur on the microsecond (μs)-second (s) timescale [3,4]. Early NMR experiments performed on BPTI clearly indicated that the phenylalanine rings present in the molecule undergo rotations, which are coupled to motion in the rest of the protein [5]. Fast dynamics (in the ps-nanosecond (ns)) occurring in each of the states affects the thermodynamics of these processes by affecting the entropy. For example several intrinsically disordered proteins (undergoing fast conformational dynamics) become ordered and fold into a stable tertiary structure upon binding their target [6]. An example of this kind of coupled folding and binding is the pKID domain of the CREB transcription factor, which is disordered when Journal of the Indian Institute of Science VOL 90:1 Jan–Mar 2010 journal.library. iisc.ernet. in 55 REVIEW Pramodh Vallurupalli it is free in solution, but adopts a structure consisting of two helices when it binds the KIX domain of the CBP protein [7]. The entropic penalty during the disorder-order transition is compensated by favorable enthalpic interactions. Here the induced fit disorder-order transition reduces the affinity of pKID for KIX but leads to a complex with high specificity. Low affinity and high specificity means the protein has a low chance of associating with the wrong partner and can readily dissociate after the signaling event is complete [6]. This type of disorder-order induced-fit is not limited to proteinprotein complexes but has been observed even in protein-RNA complexes where either the protein or the RNA or both are disordered when they are free and fold upon complex formation [8]. Rather than folding upon binding, the opposite unfolding for binding is observed when talin binds vinculin. Here part of the talin protein unfolds when it forms a complex with vinculin [9]. Any biomolecular conformational dynamics occurring on or slower than the timescale of the processes of interest affects the kinetics of the processes. As proteins and nucleic acids move on a complex energy surface [2] due to the large number of protein and solvent degrees of freedom, the molecule can populate conformations other than the ground state as illustrated in figure 1a using a simplified one-dimensional energy surface. When the energy difference between the ground (major) state and excited (minor) state is 5kBT (kB is the Boltzman constant and T is the absolute temperature) the population of the excited state will still be ∼0.7% of the ground state population. In the case of the hammerhead ribozyme it is not the ground state but such a transiently populated excited state formed by significant conformational rearrangements of the ground state structure that is the active conformation. Thus the rate of exchange between the ground state and the active excited state affects the rate of catalysis [10]. Similarly NMR experiments have demonstrated that the SH3 domain from the Fyn tyrosine kinase (Fyn SH3 domain) folds via a transiently populated intermediate [11]. These examples show that it is not enough to think of proteins and nucleic acids as static structures, but as an ensemble of interconverting conformations. Sometimes as in the case of the hammerhead ribozyme it is such a transiently formed excited state with a low population that is important for function. Single molecule experiments have also clearly shown that the conformational dynamics of enzymes affects their rate of catalysis [12]. Just as the structures of the predominantly populated ground states of proteins and nucleic acids have aided in understanding their function, it is necessary to Figure 1: (a) A protein undergoing exchange between two states A and B. A is the ground (major) state and B is the excited state (minor) state. In addition to the change in structure there can be a change in the local dynamics. This is illustrated for a valine side chain (shown in yellow), which becomes more flexible in the excited state (S2A > S 2 B). The relationship between the populations of the two states and forward and reverse rate constants is also shown. (b) Calculated N NMR spectrum (of a site in the protein) for various values of kex . Here the difference in chemical shifts between the two states = B − A = 5 ppm, the field strength is 800 MHz and the minor state population pb = 0.07. The difference in the resonance frequency of the nucleus between the two states A and B ω (rad/s), scales with the field strength. 56 Journal of the Indian Institute of Science VOL 90:1 Jan–Mar 2010 journal.library. iisc.ernet. in Structure and dynamics of protein excited states with millisecond lifetimes REVIEW characterize the dynamics and obtain structures of these transiently formed excited states to understand processes like the ones mentioned above. NMR spectroscopy is a particularly powerful technique to characterize the dynamics of biomolecules, as it is sensitive to motion occurring in the entire picosecond-second timescale [4,13]. Further the dynamics can be monitored at potentially every NMR active nucleus in the molecule. Thus by suitable labeling dynamics can be monitored at various hydrogen (1H or 2H), nitrogen (15N), carbon (13C), phosphorus (31P) sites in the molecule. Over the last few years there has been considerable progress in obtaining structures of excited states and ensembles of structures, which represent the conformations that biomolecules adopt in solution [14-19]. These include the use of order parameters to obtain an ensemble of structures that the molecule samples over the ps-ns timescale [14], residual dipolar couplings (RDCs) to obtain an ensemble of structures that the molecule adopts over the ps-μs timescale [15], paramagnetic relaxation enhancement (PRE) [16,17] and CarrPurcell-Meiboom-Gill (CPMG) experiments to characterize transiently formed structures [18,19]. Some of the recently developed CPMG NMR experiments to obtain structures and characterize the dynamics of the excited states of proteins with millisecond lifetimes are surveyed in this article. Characterizing excited states using CPMG experiments Since the excited states have very low populations they give rise to peaks with very low intensities in the NMR spectrum of the molecule. These weak peaks are further broadened because these excited states are transiently populated. This is illustrated in figure 1b for a 15N nucleus when the minor state (state B) is populated to 7% of the ground state (pb = 0.07). The Bloch-McConnell equations describe the evolution of magnetization under chemical exchange [20,21]. For two state exchange transverse magnetization evolves according to: