Large Domain Fluctuations on 50-ns Timescale Enable Catalytic Activity in Phosphoglycerate Kinase

Abstract

Large-scale domain motions of enzymes are often essential for their biological function. Phosphoglycerate kinase has a wide open domain structure with a hinge near the active center between the two domains. Applying neutron spin echo spectroscopy and small-angle neutron scattering we have investigated the internal domain dynamics. Structural analysis reveals that the holoprotein in solution seems to be more compact compared to the crystal structure but would not allow the functionally important phosphoryl transfer between the substrates if the protein were static. Brownian large-scale domain fluctuation dynamics on a timescale of 50 ns was revealed by neutron spin echo spectroscopy. The dynamics observed was compared to the displacement patterns of low-frequency normal modes. The displacements along the normal-mode coordinates describe our experimental results reasonably well. In particular, the domain movements facilitate a close encounter of the key residues in the active center to build the active configuration. The observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable the function of the protein. Moreover, the presence of the substrates increases the rigidity, which is deduced from a faster dynamics with smaller amplitude.

Neutron Spin-Echo and TOF Reveals Protein Dynamics in Solution

“…ADH with and without nicotinamide adenine dinucleotide cofactor (NAD) has been investigated in a series of neutron spin-echo experiments a few years ago [1,3] and subsequently the developed methodology was also applied to phosphoglycerate kinase [4,5]. The crystal structure of ADH is known (PDB: 2hcy) and was used in the computations of molecular properties.…”

Concept for a time-of-flight Small Angle Neutron Scattering instrument at the European Spallation Source

“…For even higher resolution in the low Q area (the primary beam detector) we are looking into single quantum detecting pixel detectors Timepix [21]. These devices can use either Silicon sensor covered with 6 Li converter [22] or neutron sensitive Micro--Channel--Plate [23]. The pixel size is 55x55 µm 2 and sub--pixel resolutions are achievable as well [22--23].…”

“…Polarization allows for studying materials with magnetic domains or fluxlines [2--4], as well as to separate coherent and incoherent background [5]. This separation is highly desirable especially for weakly scattering samples with important details at larger Q, so especially for biological molecules and complexes [6]. In recent years it has become more and more common to investigate samples in situ under a variety of physical conditions [7--8].…”

Exploring internal protein dynamics by neutron spin echo spectroscopy

“…13 Internal dynamics was revisited by Bu et al 14 followed by successful attempts to separate interprotein diffusion and internal dynamics. 3,6 In the following we will discuss the neutron scattering of proteins with respect to the protein structure and dynamics and how to access these by small angle neutron scattering (SANS) and NSE. Therefore we first describe, with three different sized proteins as examples (see Fig.…”

“…Examples for such proteins are alcohol dehydrogenase with a cleft opening motion, 3 hexokinase with a locking domain motion 4 or phosphoglycerate kinase with a large hinge bending motion. 5,6 There are only a few experimental techniques available that access these length and timescales like NMR 7 or fluorescence resonance energy transfer. 8 A strong point of neutron spin echo spectroscopy (NSE) compared to other non-scattering techniques is the ability to study nanosecond dynamics in the coherent scattering on different length scales by changing the scattering vector Q in a non-destructive experiment without mutation of the protein.…”