No, the RAM will 'work properly' regardless of the configuration. If you install 1GB of RAM in one slot the system will recognize and use 1GB of RAM in Single Channel Mode. If you add another 1GB stick the system will recognize and use 2GB of RAM in Dual Channel Mode. If you add a 3rd 1GB stick the system will recognize and use 3GB of RAM in Single Channel Mode.
Single channel mode uses all of the installed memory using the normal system bus bandwidth. Dual channel mode simple doubles the bandwidth. It does not affect the amount of memory installed, reported, or used.
Your motherboard manual recommends
"If only one DDR3 memory module is installed, it is recommended to install it in the DDR3_1 or DDR3_3 sockets."
That means the system may not boot with only one stick installed in Slot DDR3_2 by design. You can test this by populating DDR3_1, DDR3_3, and then DDR3_2.