Probing the Flexibility of the Bacterial Reaction Center:  The Wild-Type Protein Is More Rigid Than Two Site-Specific Mutants

Abstract

Experimental and theoretical studies have stressed the importance of flexibility for protein function. However, more local studies of protein dynamics, using temperature factors from crystallographic data or elastic models of protein mechanics, suggest that active sites are among the most rigid parts of proteins. We have used quasielastic neutron scattering to study the native reaction center protein from the purple bacterium Rhodobacter sphaeroides, over a temperature range of 4-260 K, in parallel with two nonfunctional mutants both carrying the mutations L212Glu/L213Asp --> Ala/Ala (one mutant carrying, in addition, the M249Ala --> Tyr mutation). The so-called dynamical transition temperature, Td, remains the same for the three proteins around 230 K. Below Td the mean square displacement, u2, and the dynamical structure factor, S(Q,omega), as measured respectively by backscattering and time-of-flight techniques are identical. However, we report that above Td, where anharmonicity and diffusive motions take place, the native protein is more rigid than the two nonfunctional mutants. The higher flexibility of both mutant proteins is demonstrated by either their higher u2 values or the notable quasielastic broadening of S(Q,omega) that reveals the diffusive nature of the motions involved. Remarkably, we demonstrate here that in proteins, point genetic mutations may notably affect the overall protein dynamics, and this effect can be quantified by neutron scattering. Our results suggest a new direction of investigation for further understanding of the relationship between fast dynamics and activity in proteins. Brownian dynamics simulations we have carried out are consistent with the neutron experiments, suggesting that a rigid core within the native protein is specifically softened by distant point mutations. L212Glu, which is systematically conserved in all photosynthetic bacteria, seems to be one of the key residues that exerts a distant control over the rigidity of the core of the protein.

Protein and solvent dynamics of the water-soluble chlorophyll-binding protein (WSCP)

“…As a complicating factor for such studies, a significant solvent contribution to the overall scattering intensity can be expected as observed before for the antenna complex LHC II [15] and for the bacterial reaction center [16,17]. In the present study we address this problem by QENS experiments on WSCP and on the corresponding buffer solution at room temperature.…”

The functional role of protein dynamics in photosynthetic reaction centers investigated by elastic and quasielastic neutron scattering

“…Qualitatively, the observed temperature dependence of molecular dynamics of PS II is similar to that of other biomolecular systems [17,18,21,22], while specific transition temperatures and motional amplitudes differ. Later, QENS studies were reported for the bacterial RC of Rhodobacter sphaeroides and of two of its site-specific mutants [16]. The data reveal a dynamical transition at about 230 K, i.e.…”

“…So far, ENS and QENS have been used to study the temperature and hydration dependence of molecular dynamics in PS II membranes of green plants [10][11][12][13][14] as well as that of bacterial RCs [15,16]. This review provides a short overview about the results of these studies.…”

Chemical proprieties of the iron-quinone complex in mutated reaction centers of Rb. sphaeroides

“…The growing conditions and the BRC purification have previously been described [9]. We used 900 nmol, 800 nmol and 1400 nmol in the WT, AA and AATyr BRCs, respectively.…”

“…sphaeroides (see Fig. 1) [8,9]. The double mutant, called later in this paper AA, has two point mutations near the Q B acidic cluster: (L212Glu/L213Asp -> Ala/Ala).…”