Planes, trains, ships, and automobiles — all machines that transport goods and materials from where they are found or made to where they are needed and used. Much the way these modern transport machines are essential to complex trade around the world, the biological world relies on transport machines to move materials and goods at the cellular level. Nanoscale transport machinery has evolved the ability to pick up and carry biological cargo, such as newly synthesized proteins, from places in the cell where proteins are made to cellular sites where the proteins must function.  The image on the cover of this issue of the Biophysical Journal depicts a cellular transport machine in chloroplasts, a chloroplast signal recognition particle (cpSRP).  Its job is to bind newly made components of the light harvesting complexes and direct them to the thylakoid membrane where they are assembled with chlorophyll to efficiently capture light energy for photosynthesis.

Understanding how these nanoscale machines operate may make it possible to design bioinspired machines that can be used to build or repair artificial solar energy conversion devices.  But ”seeing” a nanoscale machine operate presents a tremendous challenge.  We have used a wide array of techniques including bioinformatics, molecular dynamics, Small Angle X-ray Scattering (SAXS), and single-molecule Fluorescence Resonance Energy Transfer (smFRET)  to produce ”movies” of how one of the components of this molecular machine – the protein cpSRP54, a 54 kDa subunit of cpSRP – moves during its operation. Bioinformatics and molecular dynamics use structural data from similar proteins and physical theories to predict the multiple structures that the protein can adopt and how they interconvert between each other. These structural predictions are then tested using SAXS and smFRET experiments. The idea of smFRET — also depicted on the cover —employs the use of two fluorescent dyes placed at distinct sites on cpSRP54, shown in red and green. By exciting only the green dye of a single molecule that is within the confocal volume of a microscope (which is a tightly focused beam of laser light in an hourglass shape, as seen in the cover image), it is possible to transfer some of this energy to the red dye molecule on that same molecule if the two dyes are in close proximity. In fact, we can quantify exactly how much energy is transferred by measuring the brightness of the green versus red colors emitted. If there is more green light emitted, the dyes are far apart and if there is more red light emitted, the dyes are close together. This allows us to accurately measure the distance between the dye attachment sites on the single protein molecule, which is then compared to the structural predictions that we obtained from the other techniques. These data were combined and used to produce the model of cpSRP54 shown in the center of the confocal volume of the cover image.  Furthermore, since smFRET is measured at the single-molecule level, it is possible to directly show that each protein can adopt a range of different structures, which all depend on what else the protein is interacting with during the transport process. The findings show that cpSRP54 goes through multiple structural states that can interconvert between each other – a finding that is supported by the SAXS data. Importantly, the model is predictive of structural states required to load cpSRP with LHC cargo and deliver it to the chloroplast thylakoid membrane.  A small site-specific mutation predicted from the model to adversely affect the transport activity of cpSRP54 was verified in functional assays, providing a high degree of confidence in our structural models.

—Rory Henderson, Feng Gao, Srinivas Jayanthi, Alicia Kight, Priyanka Sharma, Robyn Goforth, Colin Heyes, Ralph Henry, Thallapuranam Suresh Kumar