TELSAM polymers are powerful crystallization chaperones meriting continued investigation (Diversity Supplement) - There is a critical need for new protein crystallization methods that require less labor, time, and resources. Pre-
viously, crystals of 10 out of 11 target proteins were readily generated by fusing them to TELSAM, a polymer-
forming crystallization chaperone. There is great need for continued investigation of TELSAM due to its potential
as a general-use protein crystallization chaperone. Lack of straightforward methods to successfully crystallize
any protein of interest significantly hinders study of molecular disease mechanisms and the development of
effective treatments. The lack of effective treatments for many diseases forces them to be addressed instead
with costly symptom management programs. The long-term goal of this project is to develop protein crystalliza-
tion methods that can result in well-ordered protein crystals on a time scale of less than a month, cost as little as
$1000 per structure, and are successful for greater than 70% of proteins of interest. The overall objective of this
proposal is to convincingly demonstrate the benefits of using TELSAM as a protein crystallization chaperone and
to clearly define the requirements for doing so. The central hypothesis is that TELSAM will accelerate the speed
and success rate of crystallization across a wide range of proteins of interest and that flexible fusion of target
proteins to the 1TEL variant will be optimal. The rationale is that TELSAM has shown great promise in preliminary
studies and has the potential to 1) decrease the cost of determining an atomic-scale protein structure, 2) accel-
erate the rate that protein structures can be determined, and 3) increase the success rate of crystallization,
expanding the range of proteins that can be structurally characterized in this way. The central hypothesis will be
tested, and the overall objective achieved by executing 2 specific aims: 1) Compare the ease of obtaining well-
ordered crystals across a range of proteins of interest with and without fusion to TELSAM. 2) Establish best
practices for successfully using TELSAM. In Aim 1, a panel of target proteins or protein complexes of varying
sizes will be crystallized alone or as flexible fusions to TELSAM. In Aim 2, selected target proteins will be flexibly
or rigidly fused to TELSAM with varying degrees of target protein loading along the polymer. Longer linker lengths
and unusually low protein concentrations in crystallization experiments will also be investigated. The proposed
research is innovative, in the applicant’s opinion, because it proposes: 1) Systematic investigation of the factors
required by TELSAM-target fusions to reliably form well-ordered crystals, 2) Investigation of 1TEL, which pre-
sents 6 copies of the target protein per turn of the TELSAM polymer and precludes any direct inter-TELSAM
contacts, 3) Investigation of semi-rigid fusions of target proteins to TELSAM, 4) Testing the limits of TELSAM-
mediated protein crystallization with target protein complexes and ligand-bound targets. The proposed research
is significant because it will enable the successful crystallization and structure determination of a greater number
and variety of biotechnology and disease-relevant proteins, ultimately leading to new biotechnology tools, more
effective disease treatments, and reduced healthcare costs.
