The 1.6 Å resolution crystal structure of a mutant plastocyanin bearing a 21–25 engineered disulfide bridge

Abstract

Plastocyanin is an electron-transfer protein which has been largely used for biophysical studies as well as for protein-engineering experiments. A surface disul®de bridge has been engineered in poplar plastocyanin to allow protein chemisorption on gold substrates. The mutated plastocyanin crystal structure has been studied at 1.6 A Ê resolution (R factor = 0.145, R free = 0.205) to characterize the effects of the engineered disul®de on the overall protein structure and on the Cu-coordination sphere in view of biophysical applications. The new orthorhombic crystal form isolated for the mutated plastocyanin displays two protein molecules per asymmetric unit.

A Combined Atomic Force Microscopy and Molecular Dynamics Simulation Study on a Plastocyanin Mutant Chemisorbed on a Gold Surface

“…An estimate for the true lateral size of the imaged object can be obtained as follows:28 for a sherical object and a tip with radius of curvature r , the real diameter d is given approximately by d=W 2 /8 r , where W is the measured apparent width. In our experiment the nominal tip radius is 20 nm and the average apparent width is W =25 nm, so that we obtain for the lateral size of adsorbed PCSS the value d =3.9 nm, which is close to crystallographic data 11. Indeed, on the basis of the graphical representation of PCSS anchored to gold by one or both sulfur atoms of the disulfide bridge (Figure 1), one expects from the crystallographic data lateral dimensions of 3.8×2.5 nm 11…”

“…Design, expression and purification of PCSS were carried out as previously reported 9. The protein integrity and the copper‐site properties of PCSS were assessed by extensive spectroscopic and structural characterisation 9, 11. The redox functionality of PCSS immobilised on a gold electrode was assessed by preliminary cyclic voltammetry experiments 10…”

“…The MD simulations were performed with CHARMM12 version c27 using PARM27 all‐atom parameter set 13. Initial coordinates of PCSS were taken from the X‐ray structure, at 0.16 nm resolution, of the mutant poplar PC bearing a disulfide bridge introduced by replacing Ile21 and Glu25 by two cysteine residues (1JXG entry of Brookhaven Protein Data Bank) 11…”

Tip to substrate distances in STM imaging of biomolecules

“…A reduced apparent height is a quite common feature displayed by biomolecules when imaged by STM [12,19,20,[22][23][24][25], and might be attributed to their poorer conductivity as compared to the metallic substrate. However, the lateral size is well reproduced in agreement with the crystallographic data [52] and also with that reported by several authors on different biological samples [12,20,23,24]. It should be remarked that since we engaged at a tip-substrate distance of about 3 nm, and by considering a tip retraction of 0.5 nm, it is evident that the tip has not come into contact with the protein molecule.…”

“…5(a)). This value is significantly lower than the physical height of the protein (around 3 nm [52]). A reduced apparent height is a quite common feature displayed by biomolecules when imaged by STM [12,19,20,[22][23][24][25], and might be attributed to their poorer conductivity as compared to the metallic substrate.…”