Recent Advances in the Understanding of the Influence of Electric and Magnetic Fields on Protein Crystal Growth

In this contribution we use nonconventional methods that help to increase the success rate of a protein crystal growth, and consequently of structural projects using X-ray diffraction techniques. In order to achieve this purpose, this contribution presents new approaches involving more sophisticated techniques of protein crystallization, not just for growing protein crystals of different sizes by using electric fields, but also for controlling crystal size and orientation. This latter was possible through the use of magnetic fields that allow to obtain protein crystals suitable for both high-resolution X-ray and neutron diffraction crystallography where big crystals are required. This contribution discusses some pros, cons and realities of the role of electromagnetic fields in protein crystallization research, and their effect on protein crystal contacts. Additionally, we discuss the importance of room and low temperatures during data collection. Finally, we also discuss the effect of applying a rather strong magnetic field of 16.5 T, for shorts and long periods of time, on protein crystal growth, and on the 3D structure of two model proteins

Electrostatic surface view of PCNA from shrimp <i>Litopenaeus vannamei</i> and human.

<p>The positively charged regions are colored in blue and negatively charged regions in red. The central channel is identified as a highly positive charged hole where the double-strand DNA can slide through it.</p

Model of <i>Lv</i>PCNA bound to WSSV PIPbox-peptide.

<p>In all figures the peptide was shorten to the consensus sequence GQHKILYYDIE that makes contact with <i>Lv</i>PCNA. Panel A shows a LigPlot where the peptide interacts with <i>Lv</i>PCNA through polar contacts (green dotted lines) and hydrophobic interaction (). Panel B shows a cartoon of the peptide (yellow) posed on a <i>Lv</i>PCNA monomer (blue), in red are identified the three region that participate in protein-protein interaction. In panel C, a surface image of <i>Lv</i>PCNA shows the hydrophobic pocket where the WSSV PIPbox-peptide (yellow) is attached. In panel D, residues that participate in <i>Lv</i>PCNA-peptide complex are tagged, side chains of residues from IDCL, Central Loop and C- terminal are green colored and the peptide residues are yellow colored.</p

Aligment of best-scoring models of PCNA-WSSV PIP box complex from docking.

<p>The model shows the final seven poses for the PIP-box peptide (cartoon) docked into the binding site of <i>Lv</i>PCNA (surface representation). Tagged residues are from PCNA and form the cavity for peptide interaction. Side chains of the consensus PIP-box residues are shown as gray lines.</p

Data reduction and refinement statistics from <i>Lv</i>PCNA structure.

<p>Values in parenthesis represent the statistics at the highest resolution bin.</p>§<p>R<i>_meas</i> is a redundancy-independent version of R<i>_symm</i>, R<i>_meas</i> = ∑<i>_h</i> √n<i>_h</i>/n<i>_h</i>−1 ∑^nh_i|Î_h−I_h,i|/∑<i>_h</i> ∑^nh_iI_h,i, where Î_h = 1/n_h ∑^nh_iI_h,i.</p

Crystal structure of <i>Litopenaues vannamei</i> PCNA.

<p>The PCNA molecule is arranged as homotrimer and each monomer is shown in different color. The most important parts for protein-protein interaction of each monomer: Interdomain Conecting Loop (IDCL), Central Loop and C-terminal are labeled.</p

Proposed interaction between LvPCNA and WSSV DNA polymerase.

<p>Lateral and frontal view of the theoretical model of the polymerase is depicted with DNA. The PIP box is shown in red. PCNA is shown as an orange ring.</p