  Electron paramagnetic resonance spectroscopy in combination with
site-directed spin-labeling is a very powerful tool for elucidating the
structure and organization of biomolecules. Gd$^{3+}$ complexes have recently
emerged as a new class of spin labels for distance determination by pulsed EPR
spectroscopy at Q- and W-band. We present CW EPR measurements at 240 GHz (8.6
Tesla) on a series of Gd-rulers of the type Gd-PyMTA---spacer---Gd-PyMTA, with
Gd-Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these
Gd-rulers show that significant dipolar broadening of the central
$|-1/2\rangle\rightarrow|1/2\rangle$ transition occurs at 30 K for Gd-Gd
distances up to $\sim$ 3.4 nm with Gd-PyMTA as the spin label. This represents
a significant extension for distances accessible by CW EPR, as nitroxide-based
spin labels at X-band frequencies can typically only access distances up to
$\sim$ 2 nm. We show that this broadening persists at biologically relevant
temperatures above 200 K, and that this method is further extendable up to room
temperature by immobilizing the sample in glassy trehalose. We show that the
peak-to-peak broadening of the central transition follows the expected $~1/r^3$
dependence for the electron-electron dipolar interaction, from cryogenic
temperatures up to room temperature. A simple procedure for simulating the
dependence of the lineshape on interspin distance is presented, in which the
broadening of the central transition is modeled as an $S=1/2$ spin whose CW EPR
lineshape is broadened through electron-electron dipolar interactions with a
neighboring $S=7/2$ spin.

Biological Physics;Quantitative Methods